Detection method

ABSTRACT

Provided herein are materials and methods for detecting colorectal neoplasms and colon cancer based on the expression levels of stool-derived eukaryotic RNA biomarkers in eukaryotic nucleic acid present in a stool sample from a subject, for example, a patient. The methods can be used for the detection of high-risk adenomas and colorectal neoplasm molecular subtypes.

FIELD OF THE INVENTION

The present invention relates to the extraction of eukaryotic nucleicacids from stool samples and the use of the nucleic acids for diagnosisand treatment of intestinal disease.

BACKGROUND

Gastrointestinal disorders, for example gastrointestinal cancer andother digestive diseases such as ulcerative colitis, irritable bowelsyndrome, and Crohn's disease, are widespread. In the US,gastrointestinal disorders are estimated to affect 60 to 70 millionpeople annually. For some disorders, early screening and diagnosis hasresulted in a reduction in mortality rates and improved quality of lifefor patients. However, standard methods of diagnosis, such ascolonoscopy, are invasive, time-consuming, and are associated withrelatively high costs. There is a continuing need for noninvasivemethods of diagnosing gastrointestinal disorders in both humans andanimals.

SUMMARY

Provided herein are methods of detecting colorectal neoplasia in asubject, the method comprising measuring the level of expression of 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28 or 29 stool-derived eukaryotic RNA biomarkersselected from the biomarkers listed in Table 1 or Table 2 or acombination of Table 1 and Table 2 in eukaryotic nucleic acid extractedfrom a stool sample from the subject; comparing the measured expressionlevel of the 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 stool-derived eukaryoticRNA biomarkers in the stool sample with the measured expression level ofthe 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, or 29 stool-derived eukaryotic RNAbiomarkers in a control, wherein a difference in the measured expressionlevel of the 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 stool-derived eukaryoticRNA biomarkers in the stool sample relative to the measured expressionlevel of the 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 stool-derived eukaryoticRNA biomarkers in the control indicates that the subject has colorectalneoplasia. Also provided is a method of detecting colorectal neoplasiain a subject, the method comprising: measuring the variant allelefrequency of one or more variant biomarker genes selected from thebiomarker genes listed in Table 3 in eukaryotic nucleic acid extractedfrom a stool sample from the subject; comparing the measured variantallele frequency of the one or more variant biomarker genes in the stoolsample with the measured variant allele frequency of the one or morevariant biomarker genes in a control, wherein a difference in thevariant allele frequency of the one or more variant biomarker genesrelative to the variant allele frequency of the one or more variantbiomarker genes in the control indicates that the subject has or is atrisk for colorectal cancer. Also provided is a method of detecting amolecular subtype of colorectal cancer in a subject, the methodcomprising: measuring the level of expression of two or more biomarkergenes selected from any of the colorectal neoplasm molecular subtypebiomarker genes listed in Table 4 in eukaryotic nucleic acid extractedfrom a stool sample from the subject; comparing the measured expressionlevel of the two or more colorectal neoplasm molecular subtype biomarkergenes in the biological sample with the measured expression level of thetwo or more colorectal neoplasm molecular subtype biomarker genes in acontrol, wherein a difference in the measured expression level of thetwo or more colorectal neoplasm molecular subtype biomarker genes in thebiological sample with the measured expression level of the two or morecolorectal neoplasm molecular subtype biomarker genes relative to thetwo or more colorectal neoplasm molecular subtype biomarker genes in thecontrol indicates the molecular subtype of colorectal cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features and advantages of the present invention will bemore fully disclosed in, or rendered obvious by, the following detaileddescription of the preferred embodiment of the invention, which is to beconsidered together with the accompanying drawings wherein like numbersrefer to like parts and further wherein:

FIG. 1A is an electrophoresis file run. The electrophoretic analysis wasused to check the quality of the RNA extracted based on a methoddescribed in the literature.

FIG. 1B is an electrophoresis file run. The electrophoretic analysis wasused to check the quality of the RNA extracted based on a methoddescribed herein.

FIG. 2A is an electrophoresis file run. The electrophoretic analysis wasused to check the quality of seRNA for samples that were extractedimmediately, without incubation in a stabilization buffer.

FIG. 2B is an electrophoresis file run. The electrophoretic analysis wasused to check the quality of seRNA for samples that were incubated in astabilization buffer and stored at room temperature for 24 hours priorto extraction.

FIG. 2C is an electrophoresis file run. The electrophoretic analysis wasused to check the quality of seRNA for samples that were incubated in astabilization buffer and stored at room temperature for 48 hours priorto extraction.

FIG. 3A depicts ROC analyses for various patient populations attainedduring internal validation of an SVM.

FIG. 3B depicts sensitivity of prediction for an SVM employed on anindependent test set.

FIG. 4A is a table listing the 274 colorectal neoplasm molecular subtypebiomarker genes employed in the Colorectal Cancer Subtyping Consortiumclassifier.

FIG. 4B is a table listing the 25 exemplary colorectal neoplasmmolecular subtype biomarker genes useful for identification ofcolorectal cancer subtype CMS1.

FIG. 5 is a heat map summarizing the stratification of patients bycolorectal cancer CMS (consensus molecular subtype) using the ColorectalCancer Subtyping Consortium classifier.

FIG. 6 depicts the correlation of 4 pairs of biological replicates whencomparing transcript expression of 398 genes as measured by AffymetrixHuman Transcriptome Array 2.0 and Illumina Targeted RNA Custom Panel.

FIG. 7 is a principal component analysis graph depicting hierarchicalclustering of 13 patients with colorectal cancer, adenomas, and noneoplastic findings.

FIG. 8 depicts six putative somatic variants identified in stool samplesderived from human subjects diagnosed with adenomas and colorectalcancer.

FIG. 9 is a table listing biomarkers relating to cancer, colorectalneoplasms, and/or gastrointestinal health where putative somaticvariants could be identified.

FIG. 10 is a table summarizing patient demographics and processingmetrics associated with the prospective training set, the prospectivehold out test set, the retrospective hold out test set, and the wholestudy cohort.

FIG. 11A is a flow chart of the eligible feature selection usingbootstrapping of the testing set.

FIG. 11B is a graph of the eligible features selected.

FIG. 12 is a graph of Raw GAPDH values for patients with no findings ona colonoscopy, benign polyps, low-risk adenomas, medium-risk adenomas,high-risk adenomas, and colorectal cancer.

FIG. 13 is a graph showing model performance for detection of high-riskadenomas (HRAs) based on internal cross-validation (n=154 patients).

FIG. 14 is a table with features ranked by Gini Importance.

FIG. 15A is a graph showing model performance for detection of HRAsbased on the independent hold out test set (n=110 patients) without thefecal immunochemical test (FIT) feature.

FIG. 15B is a graph showing model performance for detection of HRAsbased on the independent hold out test set (n=110 patients) with thefecal immunochemical test (FIT) feature.

FIG. 16A is a graph showing model predictions sorted by disease severitywithout the fecal immunochemical test (FIT) feature.

FIG. 16B is a graph showing model predictions sorted by disease severitywith the fecal immunochemical test (FIT) feature.

FIG. 17A is a graph showing results of an incremental downsamplinganalysis without the fecal immunochemical test (FIT) feature.

FIG. 17B is a graph showing results of an incremental downsamplinganalysis with the fecal immunochemical test (FIT) feature.

FIG. 18 is a graph showing model performance on all samples in the holdout test set, including 11 additional colorectal cancer (CRC) samples.

FIG. 19 is a graph showing model performance on all samples in the holdout test set, including 11 additional colorectal cancer (CRC) samples,extrapolated to a generalized screening population.

DETAILED DESCRIPTION

This description of preferred embodiments is intended to be read inconnection with the accompanying drawings, which are to be consideredpart of the entire written description of this invention. The drawingfigures are not necessarily to scale and certain features of theinvention may be shown exaggerated in scale or in somewhat schematicform in the interest of clarity and conciseness. In the description,relative terms such as “horizontal,” “vertical,” “up,” “down,” “top” and“bottom” as well as derivatives thereof (e.g., “horizontally,”“downwardly,” “upwardly,” etc.) should be construed to refer to theorientation as then described or as shown in the drawing figure underdiscussion. These relative terms are for convenience of description andnormally are not intended to require a particular orientation. Termsincluding “inwardly” versus “outwardly,” “longitudinal” versus “lateral”and the like are to be interpreted relative to one another or relativeto an axis of elongation, or an axis or center of rotation, asappropriate. Terms concerning attachments, coupling and the like, suchas “connected” and “interconnected,” refer to a relationship whereinstructures are secured or attached to one another either directly orindirectly through intervening structures, as well as both movable orrigid attachments or relationships, unless expressly describedotherwise. The term “operatively connected” is such an attachment,coupling or connection that allows the pertinent structures to operateas intended by virtue of that relationship. When only a single machineis illustrated, the term “machine” shall also be taken to include anycollection of machines that individually or jointly execute a set (ormultiple sets) of instructions to perform any one or more of themethodologies discussed herein. In the claims, means-plus-functionclauses, if used, are intended to cover the structures described,suggested, or rendered obvious by the written description or drawingsfor performing the recited function, including not only structuralequivalents but also equivalent structures.

The present invention is based in part on the inventors' development ofa method to separate eukaryotic cells from bacterial cells in a stoolsample, for example, a stool sample obtained from a mammal. Within thecolon, there are about approximately 1×10¹³ bacterial cells per gram ofintestinal content. This colonic microflora can include between 300-1000species. A stool or fecal sample is a complex macromolecular mixturethat includes not only eukaryotic cells sloughed off from the intestinallumen of the gastrointestinal tract, but microbes, including bacteriaand any gastrointestinal parasites, indigestible unabsorbed foodresidues, secretions from intestinal cells, and excreted material suchas mucous and pigments. Normal stool is made up of about 75% water and25% solid matter. Bacteria make up about 60% of the total dry mass offeces. The high bacterial load can contribute to an unfavorablesignal-to-noise ratio for the detection of eukaryotic biomarkers from astool sample. Furthermore, the eukaryotic signals can be heavilydegraded. Extraction and processing of such eukaryotic nucleic acids canpromote or accelerate degradation, which severely limits furtheranalysis.

The extraction method permits the isolation of high-quality eukaryoticRNA from a stool sample. The methods are described in InternationalApplication WO2018/081580, which is herein incorporated by reference inits entirety. We may refer to stool-derived eukaryotic RNA (seRNA) tospecify the eukaryotic RNA preserved during the process of fecal mattergeneration, and which is subsequently extracted from stool samples bythe method disclosed in International Application WO2018/081580.

Thus, the inventors developed materials and methods for noninvasivelyassessing the transcriptome of human colorectal cancers and colorectalneoplasia. The materials and methods disclosed herein provide efficientand sensitive detection of eukaryotic nucleic acids in a human stoolsample. The inventors have found that they could detect colorectalneoplasms based on the expression levels and variants of stool-derivedeukaryotic RNA biomarkers in eukaryotic nucleic acid present in a stoolsample from the subject. The detection methods can be configured in waysthat are useful for detecting various forms and subtypes of colorectalcancers or colorectal neoplasia.

More specifically, the materials and methods disclosed herein can beused to detect high-risk adenomas (HRAs) based on the expression levelsof stool-derived eukaryotic RNA biomarkers in eukaryotic nucleic acidpresent in a stool sample from the subject. Disclosed herein is amodel-based approach for prediction or identification of colorectalneoplasms, and specifically, high-risk adenomas. In some embodiments,the model can be based on the expression level of two or morestool-derived eukaryotic RNA biomarkers listed in Table 1 and Table 2 ineukaryotic nucleic acid present in a stool sample from the subject. Insome embodiments, the model can be based on the expression level of twoor more stool-derived eukaryotic RNA biomarkers, for example, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, or 29 of the stool-derived eukaryotic RNA biomarkersselected from the stool-derived eukaryotic RNA biomarkers listed inTable 1 or Table 2 or a combination of Table 1 and Table 2. In someembodiments, the model can be based on expression level of two or morestool-derived eukaryotic RNA biomarkers, for example 2, 3, 4, 5, 6, 7,8, 9, 10, or 11 of the stool-derived eukaryotic RNA biomarkers listed inTable 1. The model can also include demographic features, for example,the subject's age and smoking status. In some embodiments, the model canalso include the results of a fecal immunochemical test (FIT)administered to a stool sample from the subject. In some embodiments,the materials and methods disclosed herein can be used to identifymedium-risk adenomas (MRAs), low-risk adenomas (LRAs), or benign polyps.

Also provided are materials and methods for detecting colorectal cancerbased on the detection of a variant biomarker in a eukaryotic nucleicacid in a stool sample from a subject. In some embodiments the variantbiomarker can be associated with colorectal cancer tumorigenesis. Thevariant can be a variant of any of the biomarkers listed in Table 3. Avariant can be a variant in a colorectal cancer driver gene, forexample, TP53, KRAS, PIK3CA, BRAF, APC, BMP3, NDRG4, SMAD4, MLH1,CTNNB1, EGFR, BRCA1, CDKN2A, CDH1, PTEN, VEGFA, MAPK3, or NRAS.

The inventors have found that they could effectively detect geneexpression signatures associated with the consensus molecular subtypes(CMS) as defined by the Colorectal Cancer Subtyping Consortium (CRCSC)in stool-derived eukaryotic RNA. More specifically, the materials andmethods disclosed herein could be used to isolate seRNA from stoolsamples that can indicate the presence of a particular subtype ofcolorectal cancer (e.g. CMS1), as defined by the CRCSC. Of individualsdiagnosed with colorectal cancer, approximately 14% have CMS1classification. CMS1 tumors are characterized by increasedmicrosatellite instability (MSI-H), hypermutation, and immuneinfiltrate. These features are consistent with tumors in which theimmune system plays an active role in detecting and surveying the tumorsite. Patients having such tumors may benefit from targetedimmunotherapy such as immune checkpoint blockade therapy. For example,both Keytruda™ (pembrolizumab) and Opdivo™ (nivolumab) have beenapproved by the FDA for the treatment of adult and pediatric patientswith unresectable or metastatic solid tumors that are MSI-H and do notbenefit from first-line chemotherapy.

Thus, provided herein are materials and methods for determining whethera human subject with colorectal cancer has gene expression signaturesassociated with CMS1. In the context of disease monitoring, the methodcan noninvasively and selectively identify this patient population andprovide treatment guidance using seRNA. The methods can be performedefficiently and noninvasively using a stool sample rather than a bloodor biopsy sample. The methods are useful in the development of aclinical plan and method of treatment for a subject having colorectalcancer or who is at risk for colorectal cancer. In some embodiments, thetwo or more biomarkers can include combinations of 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30,35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180 or more of themarkers in FIG. 4 or Table 4. In some embodiments, the markers can becontained within differentially expressed transcript clusters and/orcommon pathways associated with colorectal cancer. Exemplary pathwaysinclude microsatellite instability (MSI), chromosomal instability (CIN),and CpG island methylator phenotype (CIMP). In some embodiments, thepathways can be cellular components pathways, cellular response tostress, stress, and RNA binding pathways.

In the context of disease monitoring, the method can noninvasively andselectively identify a patient population and provide treatmentguidance. The methods can be performed efficiently and noninvasivelyusing a stool sample rather than a blood or biopsy sample. The methodsare useful in the development of a clinical plan and method of treatmentfor a subject having colorectal neoplasms or colorectal cancer or who isat risk for colorectal neoplasms or colorectal cancer.

The methods and materials disclosed herein include methods for isolatingeukaryotic nucleic acids from a stool sample. Such eukaryotic nucleicacids can be evaluated for levels of specific biomarkers that may beindicative of a gastrointestinal disorder or disease, for example, acolorectal neoplasm or colorectal cancer, in a eukaryote, for example, amammal. The mammal can be a human or a non-human animal, for example, ahuman, dog, cat, non-human primate, ruminant, ursid, equid, pig, sheep,goat, camelid, buffalo, deer, elk, moose, mustelid, rabbit, guinea pig,hamster, rat, mouse, pachyderm, rhinoceros, or chinchilla.

The inventors have found that that they could effectively separateeukaryotic cells from bacterial cells in a eukaryotic stool sample. Theinventors have also found that they could detect eukaryotic biomarkersin the RNA isolated from such eukaryotic cells. Such biomarkers may beuseful for the detection of gastrointestinal disorders, for example,colorectal cancer, celiac disease, Crohn's disease, ulcerative colitis,gastritis, gastroenteritis, gastric cancer, gastric ulcers, necrotizingenterocolitis, gastrointestinal stromal tumors, gastrointestinallymphoma, gastrointestinal neoplasia, lymphosarcoma, adenoma,hyperplastic change, adenocarcinoma, inflammatory bowel disease,irritable bowel syndrome, pancreatic neoplasia, hepatic neoplasia,cholangiocarcinoma, colitis. Provided herein are materials and methodsfor determining whether a subject, for example, a human, a dog, or acat, is at risk for gastrointestinal disease, for example, a colorectalneoplasm, for example, a high-risk adenoma or colorectal cancer. Alsoprovided are materials and methods for diagnosis of disease and methodsof identifying the health status of a subject.

The methods and compositions disclosed herein are generally andvariously useful for the detection, diagnosis, classification, andtreatment of gastrointestinal disorders, for example a colorectalneoplasm or colorectal cancer. Methods of detection can includemeasuring the expression level in a stool sample of one, two, or morebiomarkers in a sample from a subject, for example, a patient, having agastrointestinal disorder or suspected of having a gastrointestinaldisorder and comparing the measured expression level to the measuredexpression level of one, two, or more biomarkers in a control. Adifference in the measured expression level of one, two, or morebiomarkers in a subject's sample relative to the measured expressionlevel of the one, two, or more biomarkers in a control is an indicationthat the subject has a gastrointestinal disorder. In some embodiments, adifference in the measured expression level of one, two, or morebiomarkers in a subject's sample relative to the measured expressionlevel of the one, two, or more biomarkers in a control is an indicationthat the subject, for example, a patient, is at risk for agastrointestinal disorder.

In some embodiments, methods of detection can include measuring theexpression level in a stool sample of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30 or more stool-derived eukaryotic RNA biomarkers in a sample from asubject, for example, a patient, having a gastrointestinal disorder, forexample, a colorectal neoplasm, or suspected of having agastrointestinal disorder, for example, a colorectal neoplasm, andcomparing the measured expression level to the measured expression levelof the 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more stool-derivedeukaryotic RNA biomarkers in a control. A difference in the measuredexpression level of the 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or morestool-derived eukaryotic RNA biomarkers in a subject's sample relativeto the measured expression level of the 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30 or more stool-derived eukaryotic RNA biomarkers in a control is anindication that the subject has a gastrointestinal disorder, forexample, a colorectal neoplasm. In some embodiments, a difference in themeasured expression level of the 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 ormore stool-derived eukaryotic RNA biomarkers in a subject's samplerelative to the measured expression level of the 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30 or more stool-derived eukaryotic RNA biomarkers in a controlis an indication that the subject, for example, a patient, is at riskfor a gastrointestinal disorder, for example, a colorectal neoplasm. Insome embodiments, a difference in the measured expression level of the2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30 or more stool-derived eukaryotic RNAbiomarkers in a subject's sample relative to the measured expressionlevel of the 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more stool-derivedeukaryotic RNA biomarkers in a control is an indication that thesubject, for example, a patient, is at risk for a particular type ofcolorectal neoplasia, for example, an adenoma, and more specifically, ahigh-risk adenoma. In any of the preceding embodiments, thestool-derived eukaryotic RNA biomarkers can be selected from thestool-derived eukaryotic RNA biomarkers listed in Table 1 or Table 2 ora combination of Table 1 and Table 2. Methods of detection can alsoinclude an analysis of variants of specific biomarkers.

In another embodiment, methods of detection of disease can includemeasuring the relative expression level proportion, for example, therelative ratios, of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or morestool-derived eukaryotic RNA biomarkers in a subject's stool sample andcomparing the relative proportion of these stool-derived eukaryotic RNAbiomarkers to the relative expression level proportion of the 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30 or more stool-derived eukaryotic RNAbiomarkers in a control. A difference in the measured relativeexpression level proportion of the 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30or more stool-derived eukaryotic RNA biomarkers in a subject's samplerelative to a control is an indication that the subject has agastrointestinal disease, for example, a colorectal neoplasm. In someembodiments, a difference in the measured expression level proportion ofthe 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more stool-derived eukaryoticRNA biomarkers in a subject's sample relative to the measured expressionlevel proportion of the 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or morestool-derived eukaryotic RNA biomarkers in a control is an indicationthat the subject is at risk for a gastrointestinal disorder, forexample, a colorectal neoplasm. In some embodiments, a difference in themeasured expression level proportion of the 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30 or more stool-derived eukaryotic RNA biomarkers in a subject'ssample relative to the measured expression level proportion of the 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30 or more stool-derived eukaryotic RNAbiomarkers in a control is an indication that the subject is at risk fora particular type of colorectal neoplasia, for example, an adenoma, andmore specifically, a high-risk adenoma. In any of the precedingembodiments, the stool-derived eukaryotic RNA biomarkers can be selectedfrom the stool-derived eukaryotic RNA biomarkers listed in Table 1 orTable 2 or a combination of Table 1 and Table 2. Methods of detectioncan also include an analysis of variants of specific biomarkers.

The methods can include determining the level of expression of 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30 or more stool-derived eukaryotic RNAbiomarkers in eukaryotic RNA isolated from a stool sample obtained froma subject by determining whether the levels of the 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30 or more stool-derived eukaryotic RNA biomarkers aredifferent relative to the levels of the same 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30 or more stool-derived eukaryotic RNA biomarkers in a control.Exemplary stool-derived eukaryotic RNA biomarkers are listed in Table 1and Table 2. Exemplary stool-derived eukaryotic RNA biomarkers caninclude ACY1, TNFRSF10B, DST, EGLN2, PER3, CTNNB1, ACHE, SMAD4, EDN1,ERBB2, GAPDH. ABCB1, MAPK3, VEZF1, KRAS, PTEN, CREBBP, SUZ12, CDHRS,CABLES1 AREG, SPATA2, PPARGC1A, DBP, CDH1, PDGFA, OGG1, CGN, and TCF7L2.

TABLE 1 Stool-derived eukaryotic RNA biomarkers Biomarker ExemplaryGenbank Entry ACY1 NM_000666.3 TNFRSF10B NM_003842.5 DST XM_011514826.3EGLN2 NM_080732.4 PER3 XM_024450585.1 CTNNB1 NM_001904.4 ACHE KJ425573.1SMAD4 NM_005359.5 EDN1 NM_001955.5 ERBB2 XM_024450643.1 GAPDHNM_002046.7

TABLE 2 Stool-derived eukaryotic RNA biomarkers Biomarker ExemplaryGenbank entry ABCB1 AF399931.1 MAPK3 BC013992.1 VEZF1 NM_007146.3 KRASM54968.1 PTEN KX398936.1 CREBBP U85962.3 SUZ12 NM_015355.4 CDHR5NM_021924.4 CABLES1 EF028204.1 AREG NM_001657.4 SPATA2 BC009481.2PPARGC1A HQ695733.1 DBP NM_001352.4 CDH1 NM_004360.5 PDGFA M22734.1 OGG1AF003595.1 CGN NM_020770.3 TCF7L2 CR536574.1

In some embodiments, the stool-derived eukaryotic RNA biomarkers canalso include subsets of stool-derived eukaryotic RNA biomarkers listedin Table 1 and Table 2. Some or all of the stool-derived eukaryotic RNAbiomarkers listed in Table 1 or Table 2 or a combination of Table 1 andTable 2 can form a panel. For example, some or all of the stool-derivedeukaryotic RNA biomarkers in Table 1 can form a panel (Panel A). Forexample, Panel A can include some or all of the stool-derived eukaryoticRNA biomarkers ACY1, TNFRSF10B, DST, EGLN2, PER3, CTNNB1, ACHE, SMAD4,EDN1, ERBB2, GAPDH. The compositions can include gene arrays and probesets configured for the specific detection of the panels of markersdisclosed herein. The compositions can also include kits comprising genearrays and probe sets configured for the specific detection of thepanels of markers disclosed herein. The methods can include identifyingthe presence of a variant in the nucleic acid sequence of astool-derived eukaryotic RNA biomarker, for example, the stool-derivedeukaryotic RNA biomarkers listed in Table 1 and Table 2.

Also provided are methods of detection of a variant in the nucleic acidsequence of a biomarker in a eukaryotic nucleic acid (e.g., seRNA). Avariant can be any mutation that contributes to tumor survival, tumorprogression, or tumor metastasis. We may refer to such mutations as“driver mutations” or “progressor mutations.” Such mutations can includesilent mutations, missense mutations, insertions, deletions, frameshiftmutations or nonsense mutations. The expression of any particularvariant can also be described as the “variant allele frequency” (VAF).Such variants can include variants in any of the biomarkers listed inFIG. 8, or 9. A variant can be a variant in a colorectal cancer drivergene, for example, TP53, KRAS, PIK3CA, BRAF, APC, BMP3, NDRG4, SMAD4,MLH1, CTNNB1, EGFR, BRCA1, CDKN2A, CDH1, PTEN, VEGFA, MAKP3, or NRAS.Exemplary stool-derived eukaryotic RNA variant biomarkers are listed inTable 3.

TABLE 3 Stool derived eukaryotic RNA biomarkers and variants BiomarkerExemplary Genbank Entry Exemplary Variant APC M74088.1 chr5: 112175639C > T p.R1450* NM_000038 c.C4348T KRAS M54968.1 chr12: 25398284 C > Tp.G12D NM_033360 c.G35A TP53 KX710182.1 chr17: 7577538 C > T p.R248QNM_000546 c.G743A BMP3 NM_001201.4 chr4: 81967371 C > T p.P266SNM_001201 c.C796T NDRG4 BC011795.2 chr16: 58538324 G > A p.G136RNM_020465 c.G406A SMAD4 NM_005359.5 chr18: 48591919 G > A p.R361HNM_005359 c.G1082A MLH1 U07343.1 chr3: 37067240 T > A p.V384D NM_000249c.T1151A CTNNB1 NM_001904.4 chr3: 41266137 C > T p.S45S NM_001904c.C134T EGFR NM_005228.5 chr7: 55228007 p.S492R NM_005228 c.A1474C BRCA1U14680.1 chr17: 41243770 A > C p.L1260V NM_007294 c.T3778G CDKN2AJQ694045.1 chr9: 2197112 G > A p.R80* NM_000077 c.C238T CDH1 NM_004360.5chr16: 68849598 G > A p.V501M NM_004360 c.G1501A PIK3CA NM_006218.4chr3: 178936091 G > A p.E545K NM_006218 c.G1633A PTEN KX398936.1 chr10:89692905 G > A p.R130Q NM_000314 c.G389A VEGFA NM_001171623.1 chr6:43745357 G > T p.E90D NM_001171623 c.G270T BRAF M95712.2 chr7: 140453136A > T p.V600E NM_004333 c.T1799A MAPK3 BC013992.1 chr16: 30128482 G > Cp.D300E NM_002746 c.C900G NRAS AF493919.1 chr1: 115256530 G > T p.Q61KNM_002524 c. C181A

The methods can include identifying the presence of a variant in thenucleic acid sequence of a biomarker, for example the biomarkers listedin Table 3. Some or all of the colorectal neoplasm biomarker geneslisted in Table 3 can form a panel (Panel B). In some embodiments, thecolorectal neoplasm biomarker genes listed in Table 3 can also includesubsets of colorectal neoplasm subtype biomarkers. The compositions caninclude gene arrays and probe sets configured for the specific detectionof the panels of markers disclosed herein. The compositions can alsoinclude kits comprising gene arrays and probe sets configured for thespecific detection of the panels of markers disclosed herein.

In another embodiment, methods of detection of disease can includemeasuring the relative variant allele frequency, for example, therelative ratios, of one, two, or more variants in any of the biomarkergenes listed in Table 3 in a subject's stool sample and comparing therelative variant allele frequency of these biomarkers to the variantallele frequency of one, two, or more biomarkers in a control. Adifference in the measured variant allele frequency of one, two, or morebiomarkers in a subject's sample relative to the measured variant allelefrequency in a control is an indication that the subject has agastrointestinal disease. In some embodiments, a difference in thevariant allele frequency of the one, two, or more biomarkers in asubject's sample relative to the measured variant allele frequency ofthe one, two, or more biomarkers in a control is an indication that thesubject is at risk for a gastrointestinal disorder.

Also provided are methods of detection of colorectal neoplasm molecularsubtype biomarkers. Colorectal cancer can be classified into fourdifferent molecular subtypes based on expression of particular markers.The four consensus molecular subtypes (CMS1-4) are predicted based uponthe expression of 274 genes (based upon their unique HUGO gene nameidentifiers), depicted in FIG. 4A. The random forest classifier,described by the CRCSC, uses the expression of the 274 genes as featuresto accurately identify the molecular subtype classification. The fourCMS subtypes include CMS1-4. CMS1 is associated with hypermutation andmicrosatellite instability. CMS1 tumors typically have an immuneinfiltrate. CMS1 tumors tend to have higher histopathological grade atdiagnosis and are associated with poor survival. CMS2 also referred toas the “canonical” subtype, are epithelial tumors characterized bymarked WNT and MYC signaling activation, and increased copy numberalterations and tend to be associated with long-term survival. CMS3 areepithelial tumors characterized by evident metabolic dysregulation, andmutations in KRAS, receptor tyrosine kinases, and the MAPK pathway. CMS4tumors are mesenchymal tumors characterized by transforming growthfactor-β activation, stromal invasion and angiogenesis. CMS4 tumors tendto be diagnosed at advanced stages (stages III and IV) and arecorrelated with poorer overall survival rates and poorer relapse freesurvival. Twenty-five genes (based upon their unique HUGO gene nameidentifiers) that are particularly influential in the prediction of CMS1are depicted in FIG. 4B and Table 4.

TABLE 4 Stool-derived eukaryotic RNA biomarkers for the CMS1 colorectalcancer subtype Biomarker Exemplary Genbank Entry QPRT BC010033.2 RNF43BC109028.2 TFAP2A BC017754.1 TSPAN6 BC012389.1 TRIM7 AF220032.1 GNLYBC023576.2 AXIN2 AF205888.1 FITM2 NM_001080472.4 GNG4 AF493872.1 VAV3AF067817.1 RETNLB NM_032579.2 DUSP4 BC002671.2 TNFAIP6 NM_007115.4 HOXC6CR456954.1 TRIB2 NM_021643.3 CEL NM_001807.5 GPR143 NM_000273.3 ASCL2NM_005170.2 SLC5A6 BC015631.2 GAS1 NM_002048.3 B3GNT6 NM_138706.5 CYP2B6AF182277.1 BCAT1 NM_005504.7 FAP NM_004460.5 BOC AY358328.1

The methods can include determining the level of expression of two ormore colorectal neoplasm subtype biomarkers in the human RNA isolatedfrom a stool sample obtained from a subject by determining whether thelevels of the two or more colorectal neoplasm subtype biomarker genes inthe stool sample from a subject are different relative to the levels ofthe same two or more colorectal neoplasm subtype biomarker genes in acontrol. Exemplary colorectal neoplasm subtype biomarker genes are shownin Table 4. Some or all of the colorectal neoplasm biomarker geneslisted in Table 4 can form a panel (Panel C). In some embodiments, thecolorectal neoplasm biomarker genes listed in Table 4 can also includesubsets of colorectal neoplasm subtype biomarkers. The compositions caninclude gene arrays and probe sets configured for the specific detectionof the panels of markers disclosed herein. The compositions can alsoinclude kits comprising gene arrays and probe sets configured for thespecific detection of the panels of markers disclosed herein.

In another embodiment, methods of detection of disease can includemeasuring the relative expression level proportion, for example, therelative ratios, of one, two, or more two or more colorectal neoplasmsubtype biomarkers in a subject's stool sample and comparing therelative proportion of these biomarkers to the relative expression levelproportion of one, two, or more biomarkers in a control. A difference inthe measured relative expression level proportion of one, two, or morebiomarkers in a subject's sample relative to a control can indicate themolecular subtype of colorectal cancer. In some embodiments, adifference in the measured expression level proportion of the one, two,or more biomarkers in a subject's sample relative to the measuredexpression level proportion of the one, two, or more biomarkers in acontrol is an indication that the subject may develop a particularsubtype of colorectal cancer.

Alternative methods to detect CMS1 tumors, also referred to as MSI-Htumors, can be used. Genomic variants in POLE, MLH1, MSH2, MSH6, andPMS2 implicated in DNA mismatch repair deficiencies have been used aspredictive biomarkers in clinical trials for immune checkpoint blockadetherapies. Gene expression profiles focused on expression of immuneinhibitory molecules, including PD-1, PD-L1, CTLA-4, LAG-3, and IDO, canfurther be used to predict the increased immunogenicity of themicroenvironment of MSI-H tumors and further predict the eligibility ofa patient to benefit from checkpoint immunotherapy.

Provided herein are stool-derived eukaryotic RNA biomarkers and panelsof stool-derived eukaryotic RNA biomarkers for use in diagnosis ofcolorectal neoplasms or a particular subtype precancerous lesion orcolorectal cancer. A biomarker is generally a characteristic that can beobjectively measured and quantified and used to evaluate a biologicalprocess, for example, colorectal neoplasm development, progression,remission, or recurrence. Biomarkers can take many forms including,nucleic acids, polypeptides, metabolites, or physical or physiologicalparameters.

In general, biomarkers from eukaryotic cells can include: a) a sequenceof deoxyribonucleic acid (DNA), b) a sequence of ribonucleic acid (RNA),c) a predicted sequence of amino acids, which comprise the backbone ofprotein, d) expression levels of ribonucleic acid biomarkers, e) apredicted expression level of an amino acid sequence or f) anycombination of the above. In some embodiments, a biomarker can be afragment of a larger sequence, for example, a fragment of a longer RNAsequence, a longer DNA sequence or a longer polypeptide sequence. Insome embodiments, biomarkers, such as GAPDH, ACTB or others, can be usedfor normalization of other biomarkers. In other embodiments, features,such as total RNA counts, total RNA input or others, can be used asbiomarkers or for normalization of other biomarkers.

Stool-derived eukaryotic RNA biomarkers can be quantified usingamplicons. Amplicons can contain zero, one, two, or more uniquesequences. Amplicons for the same stool-derived eukaryotic RNA biomarkercan vary in percent sequence identity. Amplicons can be designed totarget different loci. Targeted loci can include: a) geographicallysimilar loci on the same transcript from the same gene, b)geographically unique loci on the same transcript from the same gene, c)geographically unique loci on different transcripts from the same gene,or d) geographically unique loci on different transcripts from differentgenes. In some embodiments, amplicons designed to target different locican reflect structural features of a particular RNA, for example,sequence or secondary structure that might either be protected orpreferentially degraded in stool. In some embodiments, ampliconsdesigned to target different loci can reflect specific diseaseparameters, for example, in diseases in which specific alternativelyspliced transcripts are increased or decreased.

A biological sample can be a sample that contains cells or othercellular material from which nucleic acids or other analytes can beobtained. A biological sample can be a control or an experimentalsample. A biological sample can be a stool sample. The biological samplecan be obtained immediately following defecation in a toilet, on theground, into a litter box, or into a collection device. In someembodiments, the biological sample can be obtained following or during aprocedure, such as an enema, a fecal swab, or an endoscopy. Thebiological sample can be tested immediately. Alternatively, thebiological sample can be stored in a buffer prior to testing, forexample an aqueous buffer, a glycerol-based buffer, a polar solventbased buffer, an osmotic balance buffer, or other buffer sufficient forpreserving the biological sample. Additionally, or alternatively, thebiological sample can be collected and stored refrigerated, for example,at 4° C., or frozen, for example, at 0° C., −20° C., −80° C., −140° C.,or lower prior to testing. The biological sample can be stored for 1month, 2 months, 4 months, 6 months, 1 year, 2 years or more prior totesting.

The biological sample can be derived from a eukaryote, for example amammal. The mammal can be a human or a non-human animal, for example, ahuman, dog, cat, non-human primate, ruminant, ursid, equid, pig, sheep,goat, camelid, buffalo, deer, elk, moose, mustelid, rabbit, guinea pig,hamster, rat, mouse, pachyderm, rhinoceros, or chinchilla. Thus, a stoolsample can be obtained from a human or a non-human animal, for example,a human, dog, cat, non-human primate, ruminant, ursid, equid, pig,sheep, goat, camelid, buffalo, deer, elk, moose, mustelid, rabbit,guinea pig, hamster, rat, mouse, pachyderm, rhinoceros, or chinchilla.

Useful methods for isolation of nucleic acids from a biological sample,for example a stool sample, that are enriched for eukaryotic nucleicacids are provided herein. The methods can include disrupting the stoolsample with buffer. The sample can be subjected to vortexing, shaking,stirring, rotation, or other methods of agitation sufficient to dispersethe solids and the stool bacteria. The temperature at which theagitation and centrifugation steps are carried out can vary, forexample, from about 4° C. to about 20° C., from about 4° C. to about 1°C., from about 4° C. to about 10° C., from about 4° C. to about 6° C.Following disruption, the sample can be subjected to one or more roundsof centrifugation. In some embodiments, the disruption step and thecentrifugation step can be repeated one, two, three, or more additionaltimes. Commercially available reagents, for example Nuclisens® EasyMag®reagents can be used for stool disruption, washing, and cell lysis.Lysis buffer can also be used to lyse the eukaryotic cells. The lysatecan be further centrifuged at any temperature for any duration of timefor any number of times. After centrifugation, the supernatant can beused as input into an automated RNA isolation machine, for example anEasyMag® instrument. In some embodiments, the extracted nucleic acidscan be treated with DNase to degrade DNA in the solution. Other methodsof RNA purification can be used; for example, following mechanical orenzymatic cell disruption, a solid phase method can be performed such ascolumn chromatography or extraction with organic solvents, for example,phenol-chloroform or thiocyanate-phenol-chloroform extraction. In someembodiments, the nucleic acids can be extracted onto a functionalizedbead. In some embodiments, the functionalized bead can further comprisea magnetic core (“magnetic bead”). In some embodiments, thefunctionalized bead can include a surface functionalized with a chargedmoiety. The charged moiety can be selected from: amine, carboxylic acid,carboxylate, quaternary amine, sulfate, sulfonate, or phosphate.

For extraction of nucleic acids, the stool sample can be disrupted inthe presence of one or more of a buffer, a surfactant, and aribonuclease inhibitor to form a suspension. The buffer can be abiologically compatible buffer, for example, Hanks balanced saltsolution, Alsever's solution, Earle's balanced salt solution, Gey'sbalanced salt solution, Phosphate buffered saline, Puck's balanced saltsolution, Ringer's balanced salt solution, Simm's balanced saltsolution, TRIS-buffered saline, or Tyrode's balanced salt solution. Thesurfactant can be an ionic or non-ionic surfactant, for example,Tween-20, or Triton-X-100. The ribonuclease inhibitor can be solventbased, protein based, or another type of method to prevent RNAdestruction, including, for example, Protector RNase Inhibitor (Roche),RNasin® (Promega), SUPERase-In™ (Thermo Fisher Scientific), RNaseOUT™(Thermo Fisher Scientific), ANTI-RNase, Recombinant RNase Inhibitor, ora cloned RNase Inhibitor. The stool sample can be disrupted in a varietyof ways, for example by vortexing, shaking, stirring, rotating, or othermethod of agitation sufficient to disperse the solids and the stoolbacteria. In some embodiments, the stool sample can be disrupted using:coated beads, magnetic beads, or a stirring implement, such as a glassrod, a metal rod, a wooden stick, or a wooden blade.

The suspension can then be separated into a liquid portion and a solidportion. The separation can be carried out, for example, bycentrifugation, filtration, targeted probes that specifically bindeukaryotic cells, antibodies, column-based filtration, bead-basedfiltration, or chromatographic methods. The liquid portion is enrichedfor bacterial nucleic acids and can be discarded. The solid portion canbe re-suspended in a buffer either in the presence or absence of asurfactant and in the presence or absence of a ribonuclease. Theseparation step can be repeated one, two, three, four, five, six, seven,eight, or more times.

The temperature at which the disruption and separation steps are carriedout can vary, for example, from about 4° C. to about 20° C., from about4° C. to about 15° C., from about 4° C. to about 10° C., from about 4°C. to about 6° C.

The resulting pellet obtained from the separation step can be suspendedin a lysis buffer, for example, a buffer comprising a chaotropic agentand optionally a surfactant to form a lysate. In some embodiments, thechaotropic agent can be guanidium thiocyanate and the surfactant can beTriton-X-100. In some embodiments, the lysis buffer can include orexclude Tris-HCl, ethylenediaminetetraacetic acid (EDTA), sodium dodecylsulfate (SDS), Nonidet P-40, sodium deoxycholate, or dithiothreitol.

The lysate can be fractionated into a portion enriched for eukaryoticnucleic acids. The fractionation can be carried out, for example bycentrifugation, filtration, targeted probes that specifically bindeukaryotic nucleic acid, antibodies, column-based filtration, bead-basedfiltration, or chromatographic methods. In some embodiments,fractionation by centrifugation can result in the formation of a bottomlayer (a pellet), comprising cell debris, a hydrophilic middle layercomprising eukaryotic nucleic acids, and a hydrophobic top layercomprising lipids and membrane fractions. The middle layer can becollected. In some embodiments, the middle layer and the top layer canbe collected together. The middle layer can be collected through anarrow bore orifice. The narrow bore orifice can be a pipette tip or asyringe fitted with a needle. The pipette tip can be, for example, a 1uL, 5 uL, 10 uL, 20 uL, or 100 uL pipette tip. The needle can be, forexample, an 18-gauge or a 15-gauge needle.

The collected layer comprising eukaryotic nucleic acids can be subjectedto further extraction. The method of further extraction can vary.Exemplary methods include magnetic particle-based methods, column-basedmethods, filter-based methods, bead-based methods, or organicsolvent-based methods. These exemplary methods can include commerciallyavailable reagents, for example Nuclisens® EasyMag® reagents(bioMerieux).

The extracted nucleic acids can be analyzed for eukaryotic biomarkersthat are relevant to gastrointestinal disorders or gastrointestinalcells. The biomarkers can provide information on the health of anindividual, i.e., the subject. These biomarkers from eukaryotic cellscan include: a) a sequence of deoxyribonucleic acid (DNA), b) a sequenceof ribonucleic acid (RNA), c) a predicted sequence of amino acids, whichcomprise the backbone of protein, d) expression levels or proportions ofexpression levels of RNA biomarkers, e) a predicted expression level ora predicted expression level proportion of an amino acid sequence, or f)any combination of the above. Isolation of biomarkers from eukaryoticcells can allow for comparison between an experimental sample and acontrol. Isolation of these biomarkers from eukaryotic cells can providea method for detection of intestinal disease in the experimental sample.Comparison can include evaluation for: a) variation in a DNA sequence,b) variation in an RNA sequence, c) variation in the predicted aminoacid sequence, d) variation in expression levels or the variation of theproportion of expression levels of RNA biomarkers, e) variation in thepredicted expression level or variation in the prediction expressionlevel proportion of an amino acid sequence, or f) a variationconstituting any combination of the above. A variation can be determinedwhen the measured biomarker of an experimental sample is different fromthe measured biomarker in a control.

The method can include obtaining an experimental sample and a control,for example, a stool sample. The stool sample contains sloughed offeukaryotic cells that can be evaluated for biomarkers. In someembodiments, the eukaryotic cells can be enterocytes, lymphocytes,enterochromiffin-like cells, entero-endocrine cells, neuro-endocrinecells, pancreatic cells, hepatic cells, gastric cells, or other cells.The method provides a way whereby the eukaryotic cells in the stoolsample can be evaluated for eukaryotic biomarkers. The biomarkers caninclude a sequence of DNA, a sequence of RNA, a predicted sequence ofamino acids, an expression level or proportion of expression level ofRNA biomarkers, a predicted expression level or a predicted expressionlevel proportion of an amino acid sequence, or any combination of theabove. In specific embodiments, the biomarker is a stool-derivedeukaryotic RNA biomarker. In some embodiments, the evaluation stepcomprises of any type of microarray sequencing, polymerase chainreaction (PCR), nucleic acid sequencing, amplicon sequencing, molecularbarcoding, or probe-capture.

The methods and compositions are also useful for selecting a clinicalplan for an individual suffering from a gastrointestinal disorder, forexample, colorectal neoplasms or colorectal cancer. Through this method,the clinical plan can include administration of further diagnosticprocedures, for example colonoscopy. In some embodiments, the clinicalplan can include a method of treatment.

The levels of the stool-derived eukaryotic RNA biomarkers can beevaluated using a variety of methods. Expression levels can bedetermined either at the nucleic acid level, for example, the RNA level,or at the polypeptide level. RNA expression can encompass expression ofseRNA, total RNA, mRNA, tRNA, rRNA, ncRNA, smRNA, miRNA, and snoRNA.Expression at the RNA level can be measured directly or indirectly bymeasuring levels of cDNA corresponding to the relevant RNA.Alternatively, or in addition, polypeptides encoded by the RNA, RNAregulators of the genes encoding the relevant transcription factors, andlevels of the transcription factor polypeptides can also be assayed.Methods for determining gene expression at the mRNA level include, forexample, microarray analysis, serial analysis of gene expression (SAGE),RT-PCR, blotting, hybridization based on digital barcode quantificationassays, multiplex RT-PCR, droplet digital PCR (ddPCR), digital PCR(dPCR), NanoDrop spectrophotometers, RT-qPCR, qPCR, UV spectroscopy,amplicon sequencing, RNA sequencing, next-generation sequencing, lysatebased hybridization assays utilizing branched DNA signal amplificationsuch as the QuantiGene 2.0 Single Plex, and branched DNA analysismethods. Digital barcode quantification assays can include the BeadArray(Illumina), the xMAP systems (Luminex), the nCounter (NanoString), theHTG EdgeSe (High Throughput Genomics), BioMark (Fluidigm), or theWafergen microarray. Assays can include DASL (Illumina), RNA-Seq(Illumina), TruSeq (Illumina), SureSelect (Agilent), Bioanalyzer(Agilent), TaqMan (ThermoFisher), GeneReader (Qiagen), or QlAseq(Qiagen).

We may use the terms “nucleic acid” and “polynucleotide” interchangeablyto refer to both RNA and DNA, including cDNA, genomic DNA, syntheticDNA, and DNA (or RNA) containing nucleic acid analogs, any of which mayencode a polypeptide of the invention and all of which are encompassedby the invention. Polynucleotides can have essentially anythree-dimensional structure. A nucleic acid can be double-stranded orsingle-stranded (i.e., a sense strand or an antisense strand).Non-limiting examples of polynucleotides include genes, gene fragments,exons, introns, messenger RNA (mRNA) and portions thereof, transfer RNA,micro RNA, ribosomal RNA, siRNA, micro-RNA, ribozymes, cDNA, recombinantpolynucleotides, branched polynucleotides, plasmids, vectors, isolatedDNA of any sequence, isolated RNA of any sequence, nucleic acid probes,and primers, as well as nucleic acid analogs. In the context of thepresent invention, nucleic acids can encode a fragment of a biomarker,for example, stool-derived eukaryotic RNA biomarkers from any of thebiomarkers listed in Table 1 and Table 2, or variant thereof or in Table3 or a variant thereof or Table 4 or a variant thereof.

An “isolated” nucleic acid can be, for example, a DNA molecule or afragment thereof, provided that at least one of the nucleic acidsequences normally found immediately flanking that DNA molecule in agenome is removed or absent. Thus, an isolated nucleic acid includes,without limitation, a DNA molecule that exists as a separate molecule,independent of other sequences (e.g., a chemically synthesized nucleicacid, or a cDNA or genomic DNA fragment produced by the polymerase chainreaction (PCR) or restriction endonuclease treatment). An isolatednucleic acid also refers to a DNA molecule that is incorporated into avector, an autonomously replicating plasmid, a virus, or into thegenomic DNA of a prokaryote or eukaryote. In addition, an isolatednucleic acid can include an engineered nucleic acid such as a DNAmolecule that is part of a hybrid or fusion nucleic acid. A nucleic acidexisting among many (e.g., dozens, or hundreds to millions) of othernucleic acids within, for example, cDNA libraries or genomic libraries,or gel slices containing a genomic DNA restriction digest, is not anisolated nucleic acid.

Isolated nucleic acid molecules can be produced in a variety of ways.For example, polymerase chain reaction (PCR) techniques can be used toobtain an isolated nucleic acid containing a nucleotide sequencedescribed herein, including nucleotide sequences encoding a polypeptidedescribed herein. PCR can be used to amplify specific sequences from DNAas well as RNA, including sequences from total genomic DNA or totalcellular RNA. Generally, sequence information from the ends of theregion of interest or beyond is employed to design oligonucleotideprimers that are identical or similar in sequence to opposite strands ofthe template to be amplified. Various PCR strategies also are availableby which site-specific nucleotide sequence modifications can beintroduced into a template nucleic acid.

Isolated nucleic acids also can be chemically synthesized, either as asingle nucleic acid molecule (e.g., using automated DNA synthesis in the3′ to 5′ direction using phosphoramidite technology) or as a series ofoligonucleotides. For example, one or more pairs of longoligonucleotides (e.g., >50-100 nucleotides) can be synthesized thatcontain the desired sequence, with each pair containing a short segmentof complementarity (e.g., about 15 nucleotides) such that a duplex isformed when the oligonucleotide pair is annealed. DNA polymerase is usedto extend the oligonucleotides, resulting in a single, double-strandednucleic acid molecule per oligonucleotide pair, which then can beligated into a vector.

Two nucleic acids or the polypeptides they encode may be described ashaving a certain degree of identity to one another. For example, astool-derived eukaryotic RNA biomarker selected from Table 1 or Table 2or a combination of Table 1 and Table 2 or in Table 3 or Table 4 and abiologically active variant thereof may be described as exhibiting acertain degree of identity. Alignments may be assembled by locatingshort sequences in the Protein Information Research (PIR) site(http://pir.georgetown.edu), followed by analysis with the “short nearlyidentical sequences” Basic Local Alignment Search Tool (BLAST) algorithmon the NCBI website (http://www.ncbi.nlm.nih.gov/blast).

As used herein, the term “percent sequence identity” refers to thedegree of identity between any given query sequence and a subjectsequence. For example, a stool-derived eukaryotic RNA biomarker sequencelisted in Table 1 or Table 2 or a combination of Table 1 and Table 2 orin Table 3 or Table 4 can be the query sequence and a fragment of astool-derived eukaryotic RNA biomarker sequence listed in Table 1 orTable 2 or a combination of Table 1 and Table 2 or in Table 3 or Table 4can be the subject sequence. Similarly, a fragment of a stool-derivedeukaryotic RNA biomarker sequence listed in Table 1 or Table 2 or acombination of Table 1 and Table 2 or in Table 3 or Table 4 can be thequery sequence and a biologically active variant thereof can be thesubject sequence.

To determine sequence identity, a query nucleic acid or amino acidsequence can be aligned to one or more subject nucleic acid or aminoacid sequences, respectively, using a computer program, for example,ClustalW (version 1.83, default parameters), HISAT, HISAT2 or SAMTools,which allow alignments of nucleic acid or protein sequences to becarried out across their entire length (global alignment).

The nucleic acids and polypeptides described herein may be referred toas “exogenous”. The term “exogenous” indicates that the nucleic acid orpolypeptide is part of, or encoded by, a recombinant nucleic acidconstruct, or is not in its natural environment. For example, anexogenous nucleic acid can be a sequence from one species introducedinto another species, i.e., a heterologous nucleic acid. Typically, suchan exogenous nucleic acid is introduced into the other species via arecombinant nucleic acid construct. An exogenous nucleic acid can alsobe a sequence that is native to an organism and that has beenreintroduced into cells of that organism. An exogenous nucleic acid thatincludes a native sequence can often be distinguished from the nativesequence by the presence of non-natural sequences linked to theexogenous nucleic acid, e.g., non-native regulatory sequences flanking anative sequence in a recombinant nucleic acid construct. In addition,stably transformed exogenous nucleic acids typically are integrated atpositions other than the position where the native sequence is found.

Nucleic acids of the invention can include nucleic acids having anucleotide sequence of any one of the stool-derived eukaryotic RNAbiomarkers listed in Table 1 or Table 2 or a combination of Table 1 andTable 2 or in Table 3 or Table 4, or a nucleic acid sequence that is atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 99%identical to a nucleic acid sequence of any one of the stool-derivedeukaryotic RNA biomarkers listed in Table 1 or Table 2 or a combinationof Table 1 and Table 2 or in Table 3 or Table 4.

A nucleic acid, for example, an oligonucleotide (e.g., a probe or aprimer) that is specific for a target nucleic acid will hybridize to thetarget nucleic acid under suitable conditions. We may refer tohybridization or hybridizing as the process by which an oligonucleotidesingle strand anneals with a complementary strand through base pairingunder defined hybridization conditions. It is a specific, i.e.,non-random, interaction between two complementary polynucleotides.Hybridization and the strength of hybridization (i.e., the strength ofthe association between the nucleic acids) is influenced by such factorsas the degree of complementary between the nucleic acids, stringency ofthe conditions involved, and the melting temperature (Tm) of the formedhybrid. The hybridization products can be duplexes or triplexes formedwith targets in solution or on solid supports.

In some embodiments, the nucleic acids can include short nucleic acidsequences useful for analysis and quantification of the stool-derivedeukaryotic RNA biomarkers listed in Table 1 or Table 2 or a combinationof Table 1 and Table 2 or in Table 3 or Table 4. Such isolated nucleicacids can be oligonucleotide primers. In general, an oligonucleotideprimer is an oligonucleotide complementary to a target nucleotidesequence, for example, the nucleotide sequence of any of thestool-derived eukaryotic RNA biomarkers listed in Table 1 or Table 2 ora combination of Table 1 and Table 2 or in Table 3 or Table 4, that canserve as a starting point for DNA synthesis by the addition ofnucleotides to the 3′ end of the primer in the presence of a DNA or RNApolymerase. The 3′ nucleotide of the primer should generally beidentical to the target sequence at a corresponding nucleotide positionfor optimal extension and/or amplification. Primers can take many forms,including for example, peptide nucleic acid primers, locked nucleic acidprimers, unlocked nucleic acid primers, and/or phosphorothioate modifiedprimers. In some embodiments, a forward primer can be a primer that iscomplementary to the anti-sense strand of dsDNA and a reverse primer canbe a primer that is complementary to the sense-strand of dsDNA. We mayalso refer to primer pairs. In some embodiments, a 5′ target primer paircan be a primer pair that includes at least one forward primer and atleast one reverse primer that amplifies the 5′ region of a targetnucleotide sequence. In some embodiments, a 3′ target primer pair can bea primer pair at least one forward primer and at least one reverseprimer that amplifies the 3′ region of a target nucleotide sequence. Insome embodiments, the primer can include a detectable label, asdiscussed below. In some embodiments, the detectable label can be aquantifiable label.

Oligonucleotide primers provided herein are useful for amplification ofany of the stool-derived eukaryotic RNA biomarkers listed in Table 1 andTable 2 or in Table 3 or Table 4. In some embodiments, oligonucleotideprimers can be complementary to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 ormore of the stool-derived eukaryotic RNA biomarkers disclosed herein,for example, the stool-derived eukaryotic RNA biomarkers listed in Table1 and Table 2 or in Table 3 or Table 4. The primer length can varydepending upon the nucleotide base sequence and composition of theparticular nucleic acid sequence of the probe and the specific methodfor which the probe is used. In general, useful primer lengths can beabout 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30 nucleotide bases. Useful primer lengths can rangefrom 8 nucleotide bases to about 60 nucleotide bases; from about 12nucleotide bases to about 50 nucleotide bases; from about 12 nucleotidebases to about 45 nucleotide bases; from about 12 nucleotide bases toabout 40 nucleotide bases; from about 12 nucleotide bases to about 35nucleotide bases; from about 15 nucleotide bases to about 40 nucleotidebases; from about 15 nucleotide bases to about 35 nucleotide bases; fromabout 18 nucleotide bases to about 50 nucleotide bases; from about 18nucleotide bases to about 40 nucleotide bases; from about 18 nucleotidebases to about 35 nucleotide bases; from about 18 nucleotide bases toabout 30 nucleotide bases; from about 20 nucleotide bases to about 30nucleotide bases; from about 20 nucleotide bases to about 25 nucleotidebases.

Also provided are probes, that is, isolated nucleic acid fragments thatselectively bind to and are complementary to any of the stool-derivedeukaryotic RNA biomarkers listed in Table 1 and Table 2 or in Table 3 orTable 4. Probes can be oligonucleotides or polynucleotides, DNA or RNA,single- or double-stranded, and natural or modified, either in thenucleotide bases or in the backbone. Probes can be produced by a varietyof methods including chemical or enzymatic synthesis.

The probe length can vary depending upon the nucleotide base sequenceand composition of the particular nucleic acid sequence of the probe andthe specific method for which the probe is used. In general, usefulprobe lengths can be about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 50, 55, 60, 65, 70,75, 80, 85, 90, 100, 110, 120, 140, 150, 175, or 200 nucleotide bases.In general, useful probe lengths will range from about 8 to about 200nucleotide bases; from about 12 to about 175 nucleotide bases; fromabout 15 to about 150 nucleotide bases; from about 15 to about 100nucleotide bases from about 15 to about 75 nucleotide bases; from about15 to about 60 nucleotide bases; from about 20 to about 100 nucleotidebases; from about 20 to about 75 nucleotide bases; from about 20 toabout 60 nucleotide bases; from about 20 to about 50 nucleotide bases inlength. In some embodiments the probe set can comprise probes directedto 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more of the stool-derivedeukaryotic RNA biomarkers listed in Table 1 or Table 2 or a combinationof Table 1 and Table 2 or in Table 3 or Table 4.

The primers and probes disclosed herein can be detectably labeled. Alabel can be a molecular moiety or compound that can be detected or leadto a detectable response, which may be joined directly or indirectly toa nucleic acid. Direct labeling may use bonds or interactions to linklabel and probe, which includes covalent bonds, non-covalentinteractions (hydrogen bonds, hydrophobic and ionic interactions), orchelates or coordination complexes. Indirect labeling may use a bridgingmoiety or linker (e.g. antibody, oligomer, or another compound), whichis directly or indirectly labeled, which may amplify a signal. Labelsinclude any detectable moiety, e.g., radionuclide, ligand such as biotinor avidin, enzyme, enzyme substrate, reactive group, chromophore(detectable dye, particle, or bead), fluorophore, or luminescentcompound (bioluminescent, phosphorescent, or chemiluminescent label).Labels can be detectable in a homogeneous assay in which bound labeledprobe in a mixture exhibits a detectable change compared to that ofunbound labeled probe, e.g., stability or differential degradation,without requiring physical separation of bound from unbound forms.

Suitable detectable labels may include molecules that are themselvesdetectable (e.g., fluorescent moieties, electrochemical labels, metalchelates, etc.) as well as molecules that may be indirectly detected byproduction of a detectable reaction product (e.g., enzymes such ashorseradish peroxidase, alkaline phosphatase, etc.) or by a specificbinding molecule which itself may be detectable (e.g., biotin,digoxigenin, maltose, oligohistidine, 2,4-dintrobenzene, phenylarsenate,ssDNA, dsDNA, etc.). As discussed above, coupling of the one or moreligand motifs and/or ligands to the detectable label may be direct orindirect. Detection may be in situ, in vivo, in vitro on a tissuesection or in solution, etc.

In some embodiments, the methods include the use of alkaline phosphataseconjugated polynucleotide probes. When an alkaline phosphatase(AP)-conjugated polynucleotide probe is used, following sequentialaddition of an appropriate substrate such as fast blue or fast redsubstrate, AP breaks down the substrate to form a precipitate thatallows in-situ detection of the specific target RNA molecule. Alkalinephosphatase may be used with a number of substrates, e.g., fast blue,fast red, or 5-Bromo-4-chloro-3-indolyl-phosphate (BCIP).

In some embodiments, the fluorophore-conjugates probes can befluorescent dye conjugated label probes, or utilize other enzymaticapproaches besides alkaline phosphatase for a chromogenic detectionroute, such as the use of horseradish peroxidase conjugated probes withsubstrates like 3,3′-Diaminobenzidine (DAB).

The fluorescent dyes used in the conjugated label probes may typicallybe divided into families, such as fluorescein and its derivatives;rhodamine and its derivatives; cyanine and its derivatives; coumarin andits derivatives; Cascade Blue™ and its derivatives; Lucifer Yellow andits derivatives; BODIPY and its derivatives; and the like. Exemplaryfluorophores include indocarbocyanine (C3), indodicarbocyanine (C5),Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Texas Red, Pacific Blue, Oregon Green 488,Alexa Fluor®-355, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546,Alexa Fluor-555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 647,Alexa Fluor 660, Alexa Fluor 680, JOE, Lissamine, Rhodamine Green,BODIPY, fluorescein isothiocyanate (FITC), carboxy-fluorescein (FAM),phycoerythrin, rhodamine, dichlororhodamine (dRhodamine™), carboxytetramethylrhodamine (TAMRA™), carboxy-X-rhodamine (ROX™) LIZ™, VIC™,NED™, PET™, SYBR, PicoGreen, RiboGreen, and the like. Near-infrared dyesare expressly within the intended meaning of the terms fluorophore andfluorescent reporter group.

In some embodiments, levels of the eukaryotic biomarkers can be analyzedon a gene array. Microarray analysis can be performed on a customizedgene array. Alternatively, or in addition, microarray analysis can becarried out using commercially-available systems according to themanufacturer's instructions and protocols. Exemplary commercial systemsinclude Affymetrix GENECHIP® technology (ThermoFisher, Walthum, Mass.),Agilent microarray technology, the NCOUNTER® Analysis System(NanoString® Technologies, Seattle, Wash.) and the BeadArray MicroarrayTechnology (Illumina, San Diego, Calif.). Nucleic acids extracted from astool sample can be hybridized to the probes on the gene array.Probe-target hybridization can be detected by chemiluminescence todetermine the relative abundance of particular sequences. Relativeabundances of particular sequences can be normalized across a gene arrayor within a gene array.

In some embodiments, the probes and probe sets can be configured as agene array. A gene array, also known as a microarray or a gene chip, isan ordered array of nucleic acids that allows parallel analysis ofcomplex biological samples. Typically, a gene array includes probes thatare attached to a solid substrate, for example a microchip, a glassslide, or a bead. The attachment generally involves a chemical couplingresulting in a covalent bond between the substrate and the probe. Thenumber of probes in an array can vary, but each probe is fixed to aspecific addressable location on the array or microchip. In someembodiments, the probes can be about 18 nucleotide bases, about 20nucleotide bases, about 25 nucleotide bases, about 30 nucleotide bases,about 35 nucleotide bases, or about 40 nucleotide bases in length. Insome embodiments, the probe set comprises probes directed to at least 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30 or more of the stool-derived eukaryoticRNA biomarkers listed in Table 1 or Table 2 or a combination of Table 1and Table 2 or in Table 3 or Table 4. The probe sets can be incorporatedinto high-density arrays comprising 5,000, 10,000, 20,000, 50,000,100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000,900,000, 1,000,000, 2,000,000, 3,000,000, 4,000,000, 5,000,000,6,000,000, 7,000,000, 8,000,000 or more different probes.

Methods of gene array synthesis can vary. Exemplary methods includesynthesis of the probes followed by deposition onto the array surface by“spotting,” in situ synthesis, using for example, photolithography, orelectrochemistry on microelectrode arrays.

In some embodiments, the probes and probe sets can be configured as areagent, that is, a pool of nucleic acids that allows parallel analysisof complex biological samples. A reagent, can be, for example, a set ofamplification probes, a library preparation, an amplicon panel, or acapture panel. Typically, a reagent includes targeted probes that aresuspended in a solution. In some embodiments the probes are designed totarget specific regions. The probes can be configured in a way thatallows for capture of specific nucleic acids. The probes can also beconfigured to allow for amplification of a specific nucleic acid. Thenumber of probes in a reagent can vary, but each probe is designed to aspecific sequence. In some embodiments, the probes can be about 10nucleotide bases, about 15 nucleotide bases, about 20 nucleotide bases,about 25 nucleotide bases, about 30 nucleotide bases, about 35nucleotide bases, or about 40 nucleotide bases in length. In someembodiments, the probe set comprises probes directed to at least 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30 or more stool-derived eukaryotic RNAbiomarkers listed in Table 1 or Table 2 or a combination of Table 1 andTable 2 or in Table 3 or Table 4. The probe sets can be incorporatedinto high-density reagents comprising 5,000, 10,000, 20,000, 50,000,100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000,900,000, 1,000,000, 2,000,000, 3,000,000, 4,000,000, 5,000,000,6,000,000, 7,000,000, 8,000,000 or more different probes.

Methods of reagent synthesis can vary. Exemplary methods includesynthesis of nucleic acid probes followed by suspension in astabilization solution. Probe reagents can contain a unique region thatserves as a molecular identifier. The reagents can be used for suchmethods as PCR, rtPCR ddPCR, dPCR, next-generation sequencing, ampliconsequencing, RNA-se, and other methods.

Levels of the eukaryotic biomarkers can also be analyzed by DNAsequencing. DNA sequencing can be performed by sequencing methods suchas targeted sequencing, whole genome sequencing, amplicon sequencing, orexome sequencing. Sequencing methods can include: Sanger sequencing orhigh-throughput sequencing. High throughput sequencing can involvesequencing-by-synthesis, pyrosequencing, sequencing-by-ligation,real-time sequencing, nanopore sequencing, or Sanger sequencing. In someembodiments, isolated RNA can be used to generate a corresponding cDNAand the cDNA can be sequenced.

The sequencing methods described herein can be carried out in multiplexformats such that multiple different target nucleic acids aremanipulated simultaneously. In some embodiments, different targetnucleic acids can be treated in a common reaction vessel or on a surfaceof a particular substrate, enabling convenient delivery of sequencingreagents, removal of unreacted reagents, and detection of incorporationevents in a multiplex manner. In some embodiments where surface-boundtarget nucleic acids are involved, the target nucleic acids may be in anarray format. In an array format, the target nucleic acids may betypically coupled to a surface in a spatially distinguishable manner.For example, the target nucleic acids may be bound by direct covalentattachment, attachment to a bead or other particle, or associated with apolymerase or other molecule that is attached to the surface. The arraymay include a single copy of a target nucleic acid at each site (alsoreferred to as a feature) or multiple copies having the same sequencecan be present at each site or feature. Multiple copies are produced byamplification methods such as bridge amplification, ampliconamplification, PCR, or emulsion PCR.

In some embodiments, a normalization step can be used to control fornucleic acid recovery and variability between samples. In someembodiments, a defined amount of exogenous control nucleic acids can beadded (“spiked in”) to the extracted eukaryotic nucleic acids. Theexogenous control nucleic acid can be a nucleic acid having a sequencecorresponding to one or more eukaryotic or non-eukaryotic sequences, forexample, a PhiX. Alternatively, or in addition, the exogenous controlnucleic acid can have a sequence corresponding to the sequence found inanother species, for example a bacterial sequence such as a Bacillissubtilis sequence. In some embodiments, the methods can includedetermining the levels of one or more housekeeping genes. In someembodiments, the methods can include normalizing the expression levelsof biomarkers to the levels of the housekeeping genes.

The methods include the step of determining whether the measuredexpression levels of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or morestool-derived eukaryotic RNA biomarkers in an experimental sample aredifferent from the measured expression levels of the same 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30 or more stool-derived eukaryotic RNA biomarkers in acontrol. In another embodiment, the methods include the step ofdetermining whether the proportion of expression levels of 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30 or more stool-derived eukaryotic RNA biomarkersin an experimental sample are different from the proportion of measuredexpression levels of the same 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 ormore stool-derived eukaryotic RNA biomarkers in a control. A differencein the expression levels or the proportion of expression levels can bean increase or a decrease.

The compositions disclosed herein are generally and variously useful forthe detection, diagnosis and treatment of colorectal neoplasms. Methodsof detection can include measuring the expression level in a stoolsample of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more stool-derivedeukaryotic RNA biomarkers selected from the biomarkers listed in Table 1or Table 2 or a combination of Table 1 and Table 2 or in Table 3 orTable 4, and comparing the measured expression level of the 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30 or more stool-derived eukaryotic RNA biomarkersselected from the biomarkers listed in Table 1 or Table 2 or acombination of Table 1 and Table 2 or in Table 3 or Table 4, in thesample with the measured expression level of 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30 or more stool-derived eukaryotic RNA biomarkers selected from thebiomarkers listed in Table 1 or Table 2 or a combination of Table 1 andTable 2 or in Table 3 or Table 4 in a control. A difference in themeasured expression level of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or morestool-derived eukaryotic RNA biomarkers selected from the biomarkerslisted in Table 1 or Table 2 or a combination of Table 1 and Table 2 ina patient's sample relative to the measured expression level of the 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30 or more stool-derived eukaryotic RNAbiomarkers selected from the biomarkers listed in Table 1 or Table 2 ora combination of Table 1 and Table 2 in a control is an indication thatthe patient has a colorectal neoplasm, or more specifically, a high-riskadenoma. In some embodiments, a difference in the measured expressionlevel of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more stool-derivedeukaryotic RNA biomarkers selected from the biomarkers listed in Table 1or Table 2 or a combination of Table 1 and Table 2 in a patient's samplerelative to the measured expression level of 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30 or more stool-derived eukaryotic RNA biomarkers selected from thebiomarkers listed in Table 1 or Table 2 or a combination of Table 1 andTable 2 in a control is an indication that the patient is at risk for acolorectal neoplasm, or more specifically, a high-risk adenoma. Thesemethods can further include the step of identifying a subject (e.g., apatient and, more specifically, a human patient) who has a colorectalneoplasm, for example, colorectal cancer or a precancerous lesion, orwho is at risk for developing a colorectal neoplasm.

A difference in the variant allele frequency of 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 stool-derived eukaryotic RNAvariant biomarkers selected from the biomarkers listed in Table 3 in asubject's sample relative to the variant allele frequency of the 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 stool-derivedeukaryotic RNA variant biomarkers selected from the biomarkers listed inTable 3 in a control is an indication that the patient has a colorectalneoplasm. In some embodiments, a difference in the measured variantallele frequency of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, or 18 stool-derived eukaryotic RNA biomarkers selected from thebiomarkers listed in Table 3 in a patient's sample relative to themeasured variant allele frequency of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, or 18 stool-derived eukaryotic RNA biomarkersselected from the biomarkers listed in Table 3 in a control is anindication that the patient is at risk for a colorectal neoplasia. Thesemethods can further include the step of identifying a subject (e.g., apatient and, more specifically, a human patient) who has colorectalneoplasia, for example, colorectal cancer or a precancerous lesion, orwho is at risk for developing a colorectal neoplasm.

A difference in the measured expression level of 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30 or more stool-derived eukaryotic RNA biomarkers selectedfrom the colorectal neoplasm molecular subtype biomarker genes listed inFIG. 4 in a patient's sample relative to the measured expression levelof the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more stool-derivedeukaryotic RNA biomarkers selected from the colorectal neoplasmmolecular subtype biomarker genes listed in FIG. 4 in a control is anindication that the patient has a molecular subtype of colorectalcancer, for example, CMS1. In some embodiments, a difference in themeasured expression level of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 ormore stool-derived eukaryotic RNA biomarkers selected from thecolorectal neoplasm molecular subtype biomarker genes listed in FIG. 4in a patient's sample relative to the measured expression level of 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30 or more stool-derived eukaryotic RNAbiomarkers selected from the colorectal neoplasm molecular subtypebiomarker genes listed in FIG. 4 in a control is an indication that thepatient is at risk for a molecular subtype of the colorectal cancer, forexample, CMS1. These methods can further include the step of identifyinga subject (e.g., a patient and, more specifically, a human patient) whohas colorectal neoplasia, for example, colorectal cancer or aprecancerous lesion, or who is at risk for developing a colorectalneoplasm.

A colorectal neoplasm can include any form of colorectal cancer. Acolorectal neoplasm can also include a polyp, for example a precancerouslesion. Colorectal cancer typically begins as a growth, termed a polyp,in the luminal lining of the colon or rectum. Colorectal polyps aregenerally divided into two categories: adenomatous polyps and benignpolyps. Adenomatous polyps can also be called adenomas. Benign polypscan also be called hyperplastic polyps, hamartomatous polyps, orinflammatory polyps. A patient with an adenomatous polyp or multipleadenomatous polyps can be classified as having high-risk adenomas,medium-risk adenomas, or low-risk adenomas. High-risk adenomas includeadenomas with carcinoma in situ or high-grade dysplasia of any size,adenomas with greater than or equal to 25% villous growth pattern of anysize, any adenomas greater than or equal to 1.0 cm in size, or anyserrated lesion greater than or equal to 1.0 cm in size. Medium-riskadenomas include 1 or 2 non-high-risk adenomas ranging 5.0 mm to 1.0 cmin size or greater than or equal to 3 non-high-risk adenomas less than1.0 cm in size. Low-risk adenomas include 1 or 2 non-high-risk adenomasless than or equal to 5.0 mm in size. Adenomatous polyps can give riseto colorectal cancer. The most common form of colorectal cancer,adenocarcinoma, originates in the intestinal gland cells that line theinside of the colon and/or rectum. Adenocarcinomas can include tubularadenocarcinomas, which are glandular cancers on a pedunculated stalk.Adenocarcinomas can also include villous adenocarcinomas, which areglandular cancers that lie flat on the surface of the colon. Othercolorectal cancers are distinguished by their tissue of origin. Theseinclude gastrointestinal stromal tumors (GIST), which arise from theinterstitial cells of Cajal; primary colorectal lymphomas, which arisefrom hematologic cells; leiomyosarcomas, which are sarcomas arising fromconnective tissue or smooth muscle; melanomas, which arise frommelanocytes: squamous cell carcinomas which arise from stratifiedsquamous epithelial tissue and are confined to the rectum; and mucinouscarcinomas, which are epithelial cancers generally associated with poorprognosis.

Symptoms of colorectal neoplasia or colorectal cancer can include, butare not limited to, a change in bowel habits, including diarrhea orconstipation or a change in the consistency of the stool lasting longerthan four weeks, rectal bleeding or blood in the stool, persistentabdominal discomfort such as cramps, gas or pain, a feeling that thebowel does not empty completely, weakness or fatigue, and unexplainedweight loss. Patients suspected of having colorectal neoplasia orcolorectal cancer may receive peripheral blood tests, including acomplete blood count (CBC), a fecal occult blood test (FOBT), a liverfunction analysis, a fecal immunochemical test (FIT), and/or otheranalysis of certain tumor markers, for example carcinoembryonic antigen(CEA) and CA19-9. Colorectal neoplasia or colorectal cancer is oftendiagnosed based on colonoscopy. During colonoscopy, any polyps that areidentified are removed, biopsied, and analyzed to determine whether thepolyp contains colorectal cancer cells or cells that have undergone aprecancerous change. Each one of the specific cancers listed above canlook different when viewed through an endoscope. Villous adenomasmelanomas, and squamous cell carcinomas are typically flat or sessile,whereas tubular adenomas, lymphomas, leiomyosarcomas, and GIST tumorsare typically pedunculated. However, flat and sessile adenomas can bemissed by gastroenterologists during colonoscopies. Biopsy samples canbe subjected to further analysis based on genetic changes of particulargenes or microsatellite instability.

Other diagnostic methods can include, sigmoidoscopy; imaging tests, forexample, computed tomography (CT or CAT) scans; ultrasound, for exampleabdominal, endorectal or intraoperative ultrasound; or magneticresonance imaging (MRI) scans, for example endorectal MRI. Other testssuch as angiography and chest x-rays can be carried out to determinewhether a colorectal cancer has metastasized.

A variety of methods for staging colorectal cancer have been developed.The most commonly used system, the TNM system is based on threefactors: 1) the distance that the primary tumor (T) has grown into thewall of the intestine and nearby areas; 2) whether the tumor has spreadto nearby regional lymph nodes (N); 3) whether the cancer hasmetastasized to other organs (M). Other methods of staging include Dukesstaging and the Astler-Coller classification.

The TNM system provides a four-stage classification of colorectalcancer. In Stage 1 (T1) colorectal cancer, the tumor has grown into thelayers of the colon wall, but has not spread outside the colon wall orinto lymph nodes. If the cancer is part of a tubular adenoma polyp, thensimple excision is performed and the patient can continue to receiveroutine testing for future cancer development. If the cancer is highgrade or part of a flat/sessile polyp, more surgery might be requiredand larger margins will be taken; this might include partial colectomywhere a section of the colon is resected. In Stage 2 (T2) colorectalcancer, the tumor has grown into the wall of the colon and potentiallyinto nearby tissue but has not spread to nearby lymph nodes. Surgicalremoval of the tumor and a partial colectomy is generally performed.Adjunct therapy, for example, chemotherapy with agents such as5-fluorouracil, leucovorin, or capecitabine, may be administered. Suchtumors are unlikely to recur, but increased screening of the patient isgenerally needed. In Stage 3 (T3) colorectal cancer, the tumor hasspread to nearby lymph nodes, but not to other parts of the body.Surgery to remove the section of the colon and all affected lymph nodeswill be required. Chemotherapy, with agents such as 5-fluorouracil,leucovorin, oxaliplatin, or capecitabine combined with oxaliplatin istypically recommended. Radiation therapy may also be used depending onthe age of the patient and aggressive nature of the tumor. In Stage 4(T4) colorectal cancer, the tumor has spread from the colon to distantorgans through the blood. Colorectal cancer most frequently metastasizesto the liver, lungs and/or peritoneum. Surgery is unlikely to cure thesecancers and chemotherapy and or radiation are generally needed toimprove survival rates.

The methods disclosed herein are generally useful for diagnosis andtreatment of colorectal neoplasia. The expression level of 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30 or more stool-derived eukaryotic RNA biomarkers,for example a stool-derived eukaryotic RNA biomarker selected from Table1 or Table 2 or a combination of Table 1 and Table 2 or in Table 3 orTable 4, is measured in a biological sample, for example a stool samplefrom a subject. The subject can be a patient having one or more of thesymptoms described above that would indicate the patient is at risk forcolorectal cancer. The subject can also be a patient having no symptoms,but who may be at risk for colorectal neoplasia based on age (forexample, above age 50), family history, obesity, diet, alcoholconsumption, tobacco use, previous diagnosis of colorectal polyps, raceand ethnic background, inflammatory bowel disease, and geneticsyndromes, such as familial adenomatous polyposis, Gardner syndrome,Lynch syndrome, Turcot syndrome, Peutz-Jeghers syndrome, andMUTYH-associated polyposis, associated with higher risk of colorectalcancer. The methods disclosed herein are also useful for monitoring apatient who has previously been diagnosed and treated for colorectalneoplasia or colorectal cancer in order to monitor remission and detectlesion recurrence.

In some embodiments, the disease-state of a subject, that is, a human ornon-human animal patient, is determined by pathological evaluation. Forexample, in one type of disease, such as colorectal cancer, the extentof disease is classified as stage 1 (T1), stage 2 (T2), stage 3 (T3),and stage 4 (T4). The colorectal cancer can be a tubular adenocarcinoma,a villous adenocarcinoma, a gastrointestinal stromal tumor, a primarycolorectal lymphoma, a leiomysarcoma, melanoma, a squamous cellcarcinoma, or a mucinous carcinoma. In another type of disease, such asinflammatory bowel disease, the disease-state is determined by locationof the disease along the intestinal tract and histological features suchas granulomas, leukocyte infiltrates, and/or crypt abscesses. Othermethods for determining disease-state such as physician determination,physical symptoms, fecal occult blood test, a fecal immunochemical test,sigmoidoscopy, FIT-DNA, CT Colonography, or a colonoscopy can also beused in conjunction with the methods disclosed herein.

Also provided are methods of determining whether a subject is at riskfor intestinal disease. Intestinal disease can include intestinalcancer, colorectal cancer, adenomatous polyps indicative of precancerouschange, irritable bowel syndrome, necrotizing enterocolitis, ulcerativecolitis, Crohn's disease celiac disease, or other intestinal disease.The method of determining whether a subject is at risk for intestinaldisease can be determined by using the invention to detect a) a sequenceof deoxyribonucleic acid (DNA), b) a sequence of ribonucleic acid (RNA),c) a predicted amino acid sequence, which comprises the backbone ofprotein, d) expression levels of ribonucleic acid biomarkers, e)prediction in the variation of a sequence in amino acid, or f) anycombination of the above, wherein a difference between the control andthe experimental sample can indicate that the subject is at risk forintestinal disease.

The methods and compositions are also useful for selecting a clinicalplan for a subject with intestinal disease. Through this method, theclinical plan can include administration of further diagnosticprocedures. In some embodiments, the clinical plan can include a methodof treatment.

Algorithms for determining diagnosis, status, or response to treatment,for example, can be determined for particular clinical conditions. Thealgorithms used in the methods provided herein can be mathematicfunctions incorporating multiple parameters that can be quantifiedusing, without limitation, medical devices, clinical evaluation scores,or biological/chemical/physical tests of biological samples. Eachmathematic function can be a weight-adjusted expression of the levels(e.g., measured levels) of parameters determined to be relevant to aselected clinical condition. Because of the techniques involved inweighting and assessing multiple marker panels, computers withreasonable computational power can be used to analyze the data.

Thus, the method of diagnosis can include obtaining a stool sample froma patient at risk for or suspected of having a colorectal neoplasm;determining the expression of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 ormore stool-derived eukaryotic RNA biomarkers selected from thestool-derived eukaryotic RNA biomarkers listed in Table 1 or Table 2 ora combination of Table 1 and Table 2 or in Table 3 or Table 4, andproviding a test value by the machine learning algorithms thatincorporate a plurality of stool-derived eukaryotic RNA biomarkers witha predefined coefficient. Exemplary machine learning algorithms includeSupport Vector Machine, Gradient Boosting, Adaptive Boosting, RandomForest, Naive Bayes, Decision Tree, and k-Nearest Neighbors, or others.A significant change in expression of a plurality of colorectal neoplasmbiomarkers relative to the control, for example, a population of healthyindividuals, indicates an increased likelihood that the patient hascolorectal neoplasia. In some embodiments, the expression levelsmeasured in a sample are used to derive or calculate a probability or aconfidence score. This value may be derived from expression levels.Alternatively, or in addition, the value can be derived from acombination of the expression levels with other factors, for example,the patient's medical history, ethnicity, gender, age, smoking status,previous genomic results, previous histopathology results, and geneticbackground. Alternatively, or in addition, the value can be derived froma combination of the expression levels with a fecal immunochemical test(FIT). In some embodiments, the method can further comprise the step ofcommunicating the test value to the patient. This method could include,for example, visual representation of the markers, numerical output ofthe markers, or other methods of communication.

In some embodiments, a prediction for one or more patients can begenerated using a model-based approach. For example, in someembodiments, a random forest model may be configured to predict diseaseabsence, disease presence and/or disease severity in one or more groups,such as colorectal cancer, HRAs, MRAs, LRAs, benign polyps, or nofindings. In some embodiments, a validation dataset and/or a testdataset may be applied to test or refine the model. Once generated, themodel is used to predict disease absence, disease presence and/ordisease severity of one or more specific patients based on the providedinputs, such as, for example, a plurality of amplicons. Althoughspecific embodiments are discussed herein, it will be appreciated thatany suitable model could include any number of decision trees, nodes,input layers, output layers, hidden layers or other varied parameters.In some embodiments, a random forest model using a greater and/or lessernumber of decision trees, a greater and/or lesser number of eligiblefeatures, etc. may be generated.

In some embodiments, the one or more models may be generated, tested,and/or executed using a system configured for disease detection. In someembodiments, the system includes a computer system having one or moreprocessors. Each processor is connected to a communicationinfrastructure (e.g., a communications bus, cross-over bar, or network).The processor can be implemented as a central processing unit, anembedded processor or microcontroller, an application-specificintegrated circuit (ASIC), and/or any other circuit configured toexecute computer executable instructions to perform one or more steps.Processors are similar to the processor discussed above and similardescription is not repeated herein. Computer system may include adisplay interface that forwards graphics, text, and other data from thecommunication infrastructure (or from a frame buffer) for display on thedisplay unit to a user.

Computer system may also include a main memory, such as a random accessmemory (RAM), and a secondary memory. The main memory and/or thesecondary memory comprise a dynamic random access memory (DRAM). Thesecondary memory may include, for example, a hard disk drive (HDD)and/or removable storage drive, which may represent a solid statememory, an optical disk drive, a flash drive, a magnetic tape drive, orthe like. The removable storage drive reads from and/or writes to aremovable storage unit. Removable storage unit may be an optical disk,magnetic disk, floppy disk, magnetic tape, or the like. The removablestorage unit may include a computer readable storage medium havingtangibly stored therein (or embodied thereon) data and/or computerexecutable software instructions, e.g., for causing the processor(s) toperform various operations and/or one or more steps.

In alternative embodiments, secondary memory may include other devicesfor allowing computer programs or other instructions to be loaded intocomputer system. Secondary memory may include a removable storage unitand a corresponding removable storage interface, which may be similar toremovable storage drive, with its own removable storage unit. Examplesof such removable storage units include, but are not limited to,universal serial bus (USB) or flash drives, which allow software anddata to be transferred from the removable storage unit to computersystem.

Computer system may also include a communications interface (e.g.,networking interface). Communications interface allows instructions anddata to be transferred between computer system and one or moreadditional systems. Communications interface also providescommunications with other external devices. Examples of communicationsinterface may include a modem, Ethernet interface, wireless networkinterface (e.g., radio frequency, IEEE 802.11 interface, Bluetoothinterface, or the like), a Personal Computer Memory Card InternationalAssociation (PCMCIA) slot and card, or the like. Instructions and datatransferred via communications interface may be in the form of signals,which may be electronic, electromagnetic, optical, or the like that arecapable of being received by communications interface. These signals maybe provided to communications interface via a communications path (e.g.,channel), which may be implemented using wire, cable, fiber optics, atelephone line, a cellular link, a radio frequency (RF) link and othercommunication channels.

The methods and system described herein may be at least partiallyembodied in the form of computer-implemented processes and apparatus forpracticing those processes. The disclosed methods may also be at leastpartially embodied in the form of tangible, non-transitorymachine-readable storage media encoded with computer executable programcode. The media may include, for example, RAMs, ROMs, CD-ROMs, DVD-ROMs,BD-ROMs, hard disk drives, flash memories, or any other non-transitorymachine-readable storage medium, wherein, when the computer program codeis loaded into and executed by a computer, the computer becomes anapparatus for practicing the method. The methods may also be at leastpartially embodied in the form of a computer into which computer programcode is loaded and/or executed, such that, the computer becomes aspecial purpose computer for practicing the methods. When implemented ona general-purpose processor, the computer program code segmentsconfigure the processor to create specific connections, circuits, andalgorithms for implementing the methods disclosed herein.

Standard computing devices and systems can be used and implemented,e.g., suitably programmed, to perform the methods described herein,e.g., to perform the calculations needed to determine the valuesdescribed herein. Computing devices include various forms of digitalcomputers, such as laptops, desktops, mobile devices, workstations,personal digital assistants, servers, blade servers, mainframes, andother appropriate computers. In some embodiments, the computing deviceis a mobile device, such as personal digital assistant, cellulartelephone, smartphone, tablet, or other similar computing device.

In some embodiments, a computer can be used to communicate information,for example, to a healthcare professional. Information can becommunicated to a professional by making that information electronicallyavailable (e.g., in a secure manner). For example, information can beplaced on a computer database such that a health-care professional canaccess the information. In addition, information can be communicated toa hospital, clinic, or research facility serving as an agent for theprofessional. Information transferred over open networks (e.g., theinternet or e-mail) can be encrypted. Patient's gene expression data andanalysis can be stored in the cloud with encryption. The method 256-bitAES with tamper protection can be used for disk encryption; SSL protocolpreferably can ensure protection in data transit, and key managementtechnique SHA2-HMAC can allow authenticated access to the data. Othersecure data storage means can also be used.

The results of such analysis above, e.g., a probability or confidencescore derived from a combination of expression levels with otherfactors, for example, the patient's medical history, ethnicity, gender,age, smoking status, previous genomic results, previous histopathologyresults, genetic background, or a fecal immunochemical test (FIT), canbe the basis of follow-up and treatment by the attending clinician. Ifthe expression level of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or morestool-derived eukaryotic RNA biomarkers, for example a stool-derivedeukaryotic RNA biomarker selected from Table 1 or Table 2 or acombination of Table 1 and Table 2 or in Table 3 or Table 4, is notsignificantly different from the expression level of the samestool-derived eukaryotic RNA biomarker in a control, the clinician maydetermine that the patient is presently not at risk for colorectalneoplasms. Such patients can be encouraged to return in the future forrescreening. The extent to which the expression level of 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30 or more stool-derived eukaryotic RNA biomarkers, forexample a stool-derived eukaryotic RNA biomarker selected from Table 1or Table 2 or a combination of Table 1 and Table 2 or in Table 3 orTable 4, is not significantly different from the expression level of thesame stool-derived eukaryotic RNA biomarker in a control can be used todetermine the duration of time before required follow-up. In someembodiments, the clinician can recommend that the patient return forfollow-up in 1 month, 2 months, 3 months, 6 months, 1 year, 2 years, 3years, 5 years, or 10 years. The methods disclosed herein can be used tomonitor any changes in the levels of the colorectal neoplasm markersover time. A subject can be monitored for any length of time followingthe initial screening and/or diagnosis. For example, a subject can bemonitored for at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35,40, 45, 50, 55, or 60 months or more or for at least 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more years.

The methods and compositions disclosed herein are useful for selecting aclinical plan for a subject at risk for or suffering from colorectalneoplasia or colorectal cancer. The clinical plan can includeadministration of further diagnostic procedures, for example, a fecaloccult blood test, a fecal immunochemical test, or a colonoscopy toremove cancer, polyps, or precancerous lesions. In some embodiments, theclinical plan can include a method of treatment. In some embodiments,the methods include selecting a treatment for a subject having acolorectal neoplasm or colorectal cancer. If the expression level of 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30 or more of the stool-derived eukaryoticRNA biomarkers selected from Table 1 or Table 2 or a combination ofTable 1 and Table 2 or Table 3 or Table 4, is significantly differentfrom the expression level of the same 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30 or more stool-derived eukaryotic RNA biomarkers in a control, thepatient may have colorectal neoplasms or colorectal cancer. In theseinstances, further screening may be recommended, for example, increasedfrequency of screening using the methods disclosed herein, as well as afetal occult blood test, a fecal immunochemical test, and/or acolonoscopy. If the expression level of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30 or more of the stool-derived eukaryotic RNA biomarkers selected fromTable 1 or Table 2 or a combination of Table 1 and Table 2 or Table 3 orTable 4 is significantly different from the expression level of the same2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30 or more of the stool-derivedeukaryotic RNA biomarkers selected from Table 1 or Table 2 or acombination of Table 1 and Table 2 or Table 3 or Table 4 in a control,the patient may have a particular type of colorectal neoplasm, forexample, a high-risk adenoma. In some embodiments, treatment may berecommended, including, for example, a colonoscopy with removal ofpolyps, chemotherapy, immunotherapy, or surgery, such as bowelresection. Thus, the methods can be used to determine the level ofexpression of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more stool-derivedeukaryotic RNA biomarkers, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30 or more of the stool-derived eukaryotic RNA biomarkers selected fromTable 1 or Table 2 or a combination of Table 1 and Table 2 or Table 3 orTable 4 or a variant thereof and then to determine a course oftreatment. A subject, that is a patient, is effectively treated whenevera clinically beneficial result ensues. This may mean, for example, acomplete resolution of the symptoms of a disease, a decrease in theseverity of the symptoms of the disease, or a slowing of the disease'sprogression. These methods can further include the steps of a)identifying a subject (e.g., a patient and, more specifically, a humanpatient) who has colorectal neoplasia or colorectal cancer and b)providing to the subject an anticancer treatment, for example, atherapeutic agent, for example and immunotherapeutic agent, surgery, orradiation therapy. An amount of a therapeutic agent provided to thesubject that results in a complete resolution of the symptoms of adisease, a decrease in the severity of the symptoms of the disease, or aslowing of the disease's progression is considered a therapeuticallyeffective amount. The present methods may also include a monitoring stepto help optimize dosing and scheduling as well as predict outcome.Monitoring can also be used to detect the onset of drug resistance, torapidly distinguish responsive patients from nonresponsive patients orto assess recurrence of a cancer. Where there are signs of resistance ornon-responsiveness, a clinician can choose an alternative or adjunctiveagent before the tumor develops additional escape mechanisms.

The methods disclosed herein can also be used in combination withconventional methods for diagnosis and treatment of colorectal cancer.Thus, the diagnostic methods can be used along with standard diagnosticmethods for colorectal cancer. For example, the methods can be used incombination with a fecal occult blood test, a fecal immunochemical test,or a colonoscopy. The methods can also be used with other colorectalcancer markers, for example, KRAS, NRAS, BRAF, CEA, CA 19-9, p53, MSL,DCC, MSI, and MMR.

The diagnostic methods disclosed herein can also be used in combinationwith colorectal cancer treatments. Colorectal cancer treatment methodsfall into several general categories: surgery, chemotherapy, radiationtherapy, targeted therapy and immunotherapy. Surgery can includecolectomy, colostomy along with partial hepatectomy, or protectomy.Chemotherapy can be systemic chemotherapy or regional chemotherapy inwhich the chemotherapeutic agents are placed in direct proximity to anaffected organ. Exemplary chemotherapeutic agents can include5-fluorouracil, oxaliplatin or derivatives thereof, irinotecan or aderivative thereof, leucovorin, or capecitabine, mitomycin C, cisplatin,and doxorubicin. Radiation therapy can be external radiation therapy,using a machine to direct radiation toward the cancer or internalradiation therapy in which a radioactive substance is placed directlyinto or near the colorectal cancer. Targeted agents can includeanti-angiogenic agents such as bevacizumab) or EGFR inhibitor monoclonalantibody (cetuximab, panitumumab), ramuciramab (anti-VEGFR2),aflibercept, regorafenib, tripfluridine-tipiracil or a combinationthereof. Targeted agents can also be combined with standardchemotherapeutic agents. Immunotherapy can include administration ofspecific antibodies, for example anti-PD-1 antibodies, anti-PD-L-1antibodies, and time-CTLA-4 antibodies, anti-CD 27 antibodies; cancervaccines, adoptive cell therapy, oncolytic virus therapies, adjuvantimmunotherapies, and cytokine-based therapies. Exemplaryimmunotherapeutics can include Keytruda, Opdiva, and iplimumab. Othertreatment methods include stem cell transplantation, hyperthermia,photodynamic therapy, blood product donation and transfusion, or lasertreatment.

We may use the terms “increased”, “increase” or “up-regulated” togenerally mean an increase in the level of a biomarker by astatistically significant amount. In some embodiments, an increase canbe an increase of at least 10% as compared to a control, for example anincrease of at least about 20%, or at least about 30%, or at least about40%, or at least about 50%, or at least about 60%, or at least about70%, or at least about 80%, or at least about 90% or up to and includinga 100% increase or any increase between 10-100% as compared to acontrol, or at least about a 0.5-fold, or at least about a 1.0-fold, orat least about a 1.2-fold, or at least about a 1.5-fold, or at leastabout a 2-fold, or at least about a 3-fold, or at least about a 4-fold,or at least about a 5-fold or at least about a 10-fold increase, or anyincrease between 1.0-fold and 10-fold or greater as compared to acontrol.

We may use the terms “decrease”, “decreased”, “reduced”, “reduction” or“down-regulated” to refer to a decrease in the level of a eukaryoticbiomarker by a statistically significant amount. In some embodiments, adecrease can be a decrease of at least 10% as compared to a control, forexample a decrease of at least about 20%, or at least about 30%, or atleast about 40%, or at least about 50%, or at least about 60%, or atleast about 70%, or at least about 80%, or at least about 90% or up toand including a 100% decrease (i.e. absent level as compared to acontrol), or any decrease between 10-100% as compared to a control, orat least about a 0.5-fold, or at least about a 1.0-fold, or at leastabout a 1.2-fold, or at least about a 1.5-fold, or at least about a2-fold, or at least about a 3-fold, or at least about a 4-fold, or atleast about a 5-fold or at least about a 10-fold decrease, or anydecrease between 1.0-fold and 10-fold or greater as compared to acontrol.

The statistical significance of an increase in a eukaryotic biomarker ora decrease in a eukaryotic biomarker can be expressed as a p-value or aq-value. Depending upon the specific eukaryotic biomarker, p-value orq-value can be less than 0.05, less than 0.01, less than 0.005, lessthan 0.002, less than 0.001, or less than 0.0005. A q-value can be aderivative to a p-value. In some embodiments the q-value can be thep-value adjusted for the false discovery rate.

A control can be a biological sample obtained from a patient or a groupof patients. In some embodiments, the control can be a reference value.A control can be obtained from an individual, or a population ofindividuals, who have been diagnosed as healthy. Healthy individuals caninclude, for example, individuals who have tested negative in a fecalparasitic test, a fecal bacteria test, a colonoscopy, or an endoscopywithin the last year. A control can be obtained from an individual, or apopulation of individuals, who have been diagnosed as diseased. Diseasedindividuals can include, for example, individuals who have testedpositive in a fecal parasitic test, a fecal bacterial test, acolonoscopy, or an endoscopy within the last year. A control can beobtained from an individual, or a population of individuals, who hadpreviously been diagnosed with disease but are currently in remission,do not have active disease, or are not currently suffering from thedisease. A control can be obtained from an individual at one, two, ormore points in time. For example, a control can be a biological sampleobtained from a subject at an earlier point in time. A control can be astandard reference value for a particular biomarker. A standardreference value can be derived based on evaluating individuals ofsimilar age, sex, gender, body size, breed, ethnic background, orgeneral health. In some embodiments, a control can be a value or valuesderived from an algorithm.

An experimental sample can be a biological sample obtained from asubject. An experimental sample can be obtained from a subject withknown or unknown health status. In some embodiments, health status of asubject can be determined, for example, by analysis of an experimentalsample, biopsy, physical examination, laboratory findings, visualinspection, or genetic analysis. The health status of a subject that canbe determined via an experimental sample can be diseased, at risk fordisease, or healthy.

Articles of Manufacture

Also provided are kits for detecting and quantifying selectedstool-derived eukaryotic RNA biomarkers in a biological sample, forexample, a stool sample. Accordingly, packaged products (e.g., sterilecontainers containing one or more of the compositions described hereinand packaged for storage, shipment, or sale at concentrated orready-to-use concentrations) and kits, are also within the scope of theinvention. A product can include a container (e.g., a vial, jar, bottle,bag, microplate, microchip, or beads) containing one or morecompositions of the invention. In addition, an article of manufacturefurther may include, for example, packaging materials, instructions foruse, syringes, delivery devices, buffers, or other control reagents.

The kit can include a compound or agent capable of detecting RNAcorresponding to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more stool-derivedeukaryotic RNA biomarkers, for example, a stool-derived eukaryotic RNAbiomarker selected from Table 1 or Table 2 or a combination of Table 1and Table 2 or Table 3 or Table 4, in a biological sample; and astandard; and optionally one or more reagents necessary for performingdetection, quantification, or amplification. In some embodiments, thekit can include a compound or agent capable of detecting RNAcorresponding to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more stool-derivedeukaryotic RNA biomarkers, for example, a stool-derived eukaryotic RNAbiomarker selected from Table 1 or Table 2 or a combination of Table 1and Table 2 or Table 3 or Table 4, in a biological sample; and astandard; and optionally one or more reagents necessary for performingdetection, quantification, or amplification. The compounds, agents,and/or reagents can be packaged in a suitable container. The kit canfurther comprise instructions for using the kit to detect and quantifynucleic acid. The kit can also contain a control or a series of controlswhich can be assayed and compared to the test sample contained. Eachcomponent of the kit can be enclosed within an individual container andall of the various containers can be within a single package, along withinstructions for interpreting the results of the assays performed usingthe kit. In some embodiments, the kits can include primers oroligonucleotide probes specific for one or more control markers. In someembodiments, the kits include reagents specific for the quantificationof 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more stool-derived eukaryoticRNA biomarkers, for example, a stool-derived eukaryotic RNA biomarkerselected from Table 1 or Table 2 or a combination of Table 1 and Table 2or Table 3 or Table 4.

In some embodiments, the kit can include reagents specific for theseparation of eukaryotic cells from bacterial cells and other stoolcomponents and extraction of stool-derived eukaryotic RNA from apatient's, for example, a human patient's, stool sample. Thus, the kitcan include buffers, emulsion beads, silica beads, stabilizationreagents, and various filters and containers for centrifugation. The kitcan also include instructions for stool handling to minimizecontamination of samples and to ensure stability of stool-derivedeukaryotic RNA in the stool sample. The kit can also include items toensure sample preservation, for example, stabilization buffers, coolantsor heat packs. In some embodiments, the kit can include a stoolcollection device.

The product may also include a legend (e.g., a printed label or insertor other medium describing the product's use (e.g., an audio- orvideotape or computer readable medium)). The legend can be associatedwith the container (e.g., affixed to the container) and can describe themanner in which the reagents can be used. The reagents can be ready foruse (e.g., present in appropriate units), and may include one or moreadditional adjuvants, carriers, or other diluents. Alternatively, thereagents can be provided in a concentrated form with a diluent andinstructions for dilution.

EXAMPLES Example 1: Human Stool Sample Procurement

Human Stool Collection: Patients were asked to defecate into a bucketthat fit over a toilet seat and the resulting samples were stored in afreezer until they were transported to the Kharkiv National MedicalUniversity (Kharkiv, Ukraine). The stool was aliquoted into 50 mLconical tubes and stored at −80° C. The samples were shipped fromKharkiv National Medical University on dry ice to Capital Biosciences(Gaithersburg, Md.) and immediately transferred to a −80° C. freezer.From there, the samples were shipped on dry ice to BioGenerator Labs(Saint Louis, Mo.) where they were stored in a −80° C. freezer untilextraction.

Human Sample Types: Stool samples were obtained from 195 patients withcolorectal cancer (stage I-IV), 126 patients with precancerous adenomas,8 patients with benign polyps, and 125 patients with negative findingson a colonoscopy, resulting in 454 aggregate samples. Healthyindividuals were patients with no history of colorectal cancer,inflammatory bowel disease, celiac disease, irritable bowel syndrome,diarrhea within the last 20 days or any other gastrointestinal disease.Benign polyp patients provided a stool sample prior to undergoing acolonoscopy where the physician detected a polyp that was deemed to bebenign via a subsequent biopsy and histological evaluation. Diseasedindividuals were patients diagnosed with colorectal cancer orprecancerous adenomas. Colorectal cancer patients had been diagnosedwith stage I-stage IV colorectal cancer via colonoscopy and subsequentbiopsy within the last month and had not yet received any post-biopsytreatment, which can include chemotherapy, radiation, and/or surgery.Precancerous adenoma patients provided a stool sample prior toundergoing a colonoscopy where the physician detected a polyp that wasdeemed to be precancerous via a subsequent biopsy and histologicalevaluation. The healthy and benign polyp individuals were matched withadenoma and cancer patients based on gender and age brackets (50-60years, 60-70 years, 70-80 years and 80-90 years). The patients used forthis collection were consented by Capital Biosciences. The SchulmanInternal Review Board provided ethical oversight for this collection.

Example 2: Human Nucleic Acid Extraction

Total Nucleic Acid Extraction: Each stool sample was placed into a 50 mLconical tube. Approximately 1,000-25,000 mg of stool were added to eachtube. An additional 20-40 mL of solution were added to each tube. Thissolution contained a mixture of Hanks Balanced Salt Solution (HBSS)(Sigma-Aldrich) with 0.05% Tween-20 (Sigma-Aldrich) and 0.0002% RNAseInhibitor (Sigma-Aldrich). The stool was suspended into solution androtated at approximately 0-10° C. for 0-10 minutes. The solution wascentrifuged at 1000 rpm at 4° C. for 10 minutes and the supernatant wasdiscarded. Approximately 4-10 mL of EasyMag® Lysis Buffer (bioMerieux)was added to the pellet and the pellet was re-suspended into solution.The solution was centrifuged at 2500-3500 rpm at 20-25° C. for 10-15minutes. During the differential centrifugation, the solution separatedinto three layers. The bottom layer included solid cellular debris, themiddle layer was a hydrophilic layer enriched for human nucleic acid andthe top layer was a hydrophobic lipid layer. The top two layers weretransferred to a new 15 mL conical tube and the solution was againcentrifuged at 2500 rpm at 20-25° C. for 10 minutes. The result fromthis centrifugation step was separation into three layers: the bottomlayer was solid cellular debris, the middle layer was a hydrophiliclayer enriched for human nucleic acid, and the top layer was ahydrophobic lipid layer. To screen large debris from the solution, a 20uL pipette tip was placed onto a 1 mL pipette tip and 2 mL of thehydrophilic layer was pipetted from the 15 mL tube and transferred to anEasyMag® Disposable cartridge (bioMerieux). Additionally, 60 uL ofEasyMag® Magnetic Silica (bioMerieux) was added to the cartridge. Thebeads were mixed into the solution for 0.5-1 minute using a pipette. Thenucleic acids, which were bound to the beads, were eluted into a buffersolution using the Specific A Protocol according to the manufacturer'sdirections. The volume of the eluted nucleic acids was 70 uL. Thisnucleic acid solution was pipetted into a 1.5 mL tube and placed on ice.The same EasyMag® Disposable cartridges (bioMerieux) that were used inthe previous step were then reloaded with an additional 2 mL of thehydrophilic layer from the same solution in the 15 mL tube usedpreviously using the same technique to screen out large debris. Anadditional 20 uL of EasyMag® Magnetic Silica (bioMerieux) was added tothe cartridge. The beads were mixed into the solution for 0.5-1 minuteusing a pipette. As described above, the nucleic acids, which were boundto the beads, were eluted into a buffer solution using the Specific AProtocol according to the manufacturer's directions. The volume of theeluted nucleic acids was 70 uL. This nucleic acid solution was pipettedinto the original 1.5 mL tube that already contained first 70 uL eluateand the combined solution was placed on ice.

DNAse Treatment: The 140 uL solution was treated withBaseline-Zero-DNase (Epicenter) at 35-40° C. for 20-40 minutes. A 1-2 mLaliquot of EasyMag® Lysis Buffer was added to the DNAse treated solutionand the sample was transferred to a new EasyMag® Disposable cartridge.The entire solution was added to the new cartridge along with 60 uL ofEasyMag® Magnetic Silica. The nucleic acids, which were bound to thebeads, were eluted into a buffer solution using the EasyMag® GenericProtocol according to the manufacturer's directions. The volume of theeluted nucleic acids was 25 uL. This nucleic acid solution was pipettedinto a 1.5 mL tube and stored at 0-6° C.

Example 3: Measurement of Human Nucleic Acid Levels in Human StoolSamples

Extraction Results: 1-2 uL of each of the samples extracted above wasevaluated for total nucleic acid and RNA integrity using the Agilent2100 Bioanalyzer. The samples were analyzed qualitatively andquantitatively. Electrophoretic analysis was used to check the qualityof the extracted RNA. The electrophoresis file was read by comparing thebands for each sample to the bands represented by the size markers inthe RNA ladder and identifying the 18S and 28S ribosomal RNA (rRNA)bands. The rRNA bands are the two large and prominent bands around the2,000-nucleotide marker on the standardization ladder. Qualitatively,adequate banding and darker band intensities indicated that ample intactnucleic acid was available for further analysis such as microarraysequencing, polymerase chain reaction (PCR), nucleic acid sequencing,molecular barcoding, or probe-capture. The electropherogram is agraphical representation for each electrophoresis file with aquantification of the RNA Integrity Number (RIN), total RNA mass, andtotal rRNA mass. Quantitatively, the larger the RIN, the more total RNAmass, and the more total rRNA mass, the higher the likelihood a samplewould be useful for further analysis such as microarray sequencing,polymerase chain reaction (PCR), nucleic acid sequencing, molecularbarcoding, or probe-capture.

FIG. 1A is an electrophoresis file for six samples and anelectropherogram for one sample that were extracted based on a methoddescribed in the literature. FIG. 1B is an electrophoresis file for sixsamples and an electropherogram for one sample that were extractedabove. Samples extracted above resulted in larger RIN and moreeukaryotic mass. The higher quality of the seRNA extracted above wasalso demonstrated by more distinct ribosomal RNA bands (18S and 28S) andless bacterial noise, as evidenced by minimal banding below the 18Sband.

Example 4: seRNA Incubation in a Stabilization Buffer

11 samples were selected to undergo testing with a stabilization buffer.These samples were split into 5-gram aliquots, resulting in threecohorts: Cohort 1 (n=11), Cohort 2 (n=11) and Cohort 3 (n=8). Cohort 1samples were extracted immediately using the method described above.(FIG. 2A). Cohort 2 samples were incubated in a stabilization buffer andstored at room temperature for 24 hours prior to extraction using themethod described above (FIG. 2B). Cohort 3 samples were incubated in astabilization buffer and stored at room temperature for 48 hours priorto extraction using the method described above (FIG. 2C).

Following extraction, all samples (n=30) were analyzed qualitativelyusing an Agilent Bioanalyzer. Clear and distinct intact ribosomal 18Sand 28S bands are detected with isolated RNA from all samples. Theintensity of the 18S and 28S, which can be used to estimate the amountof eukaryotic RNA, increased with incubation in a stabilization buffer.Further, bacterial noise, illustrated by banding below the 18S,decreased with incubation in a stabilization buffer.

Data from the Agilent Bioanalyzer also enabled quantification of the RNAintegrity number (RIN) and eukaryotic mass. RIN was adequate for allsamples. The overall RIN of each cohort increased with incubation in astabilization buffer, with mean RINs of 4.6, 5.9, and 7.1 for Cohort 1,Cohort 2, and Cohort 3, respectively. Eukaryotic mass was adequate inall samples. The overall eukaryotic mass increased with incubation in astabilization buffer, with mean masses of 11.1 ng, 39.7 ng, and 78.4 ngfor Cohort 1, Cohort 2, and Cohort 3, respectively.

Example 5: Analysis of RNA Transcripts

330 samples were chosen for analysis using the Affymetrix GeneChip™Human Transcriptome Array 2.0 (Santa Clara, Calif.). Approximately 100ng of DNase-free fecal RNA was amplified with the Ambio WT-pico kit withsubsequent hybridization to the Affymetrix GeneChip™ Human TranscriptomeArray 2.0 as per the manufacturer's protocol. All samples werenormalized using the Signal Space Transformation-Robust MultiarrayAnalysis (SST-RMA) with the Affymetrix Expression Console™.

Of the 70,523 transcript clusters in the Affymetrix Microarray, a subsetof 5,149 transcript clusters that correspond to 3,977 genes werepreselected to evaluate for differential expression. This initialselection reduced the false discovery rate and filtered out genes thathave no known function in cancer development and progression.

The 330 individuals were split into a training set of 265 individualsand a testing set of 65 individuals. The training set was used toidentify the differentially expressed genes and build a computationalmodel, whereas the testing set was used to determine the detectionaccuracy of the computational model. The standard LIMMA package was usedto identify a subset of RNA transcript clusters which weredifferentially expressed between individuals with either precancerousadenomas or CRC and individuals with no findings on a colonoscopy. Allbiomarkers were ranked according to the log odds scores and the 200highest ranked biomarkers (p<0.05) served as the features in buildingthe machine learning model. The Support Vector Machine Model (ν-SVM)with RBF kernel was chosen for model development. The kernel functionallows for the calculation of the distance between individuals byexpanding the features into a higher dimensional space which is notexplicitly computed. SVM finds the maximum margin hyperplane thatseparates the label groups. The parameter ν defines the lower bound ofthe fraction of individuals that are used to determine the maximummargin. The SVM model was trained using expression levels for the 200transcripts from all 265 individuals in the training set. Internalvalidation of the SVM attained a total ROC AUC of 0.776. The modelattained a ROC AUC of 0.829 and 0.788 when evaluating CRC and adenomas,respectively (FIG. 3A).

This multi-target RNA biomarker algorithm was also used on the 65individuals within the independent test set. The model correctlyidentified 79% (34 out of 43) of all individuals that had positivefindings on a screening colonoscopy, 95% of individuals withprecancerous adenomas and 65% of individuals with cancer. Modelsensitivity for CRC was directly correlated with size such that 72% oftumors >4 cm in diameter were accurately detected. Model sensitivity foradenomas was agnostic to size, with 100% prediction accuracy for bothsmall (<5 mm) and large (>1 cm) lesions (FIG. 3B).

Example 6: CRC Molecular Subtyping Using seRNA Expression Signatures

Of the 70,523 transcript clusters in the Affymetrix Microarray, a subsetof transcript clusters that correspond to 274 genes was selected toannotate patient samples derived from individuals diagnosed withcolorectal cancer with a consensus molecular subtype (CMS) of CRCdefined by the Colorectal Cancer Subtyping Consortium (CRCSC) (FIG. 4A).The CRCSC classifier is organized based on the importance of each genewith regards to its ability to promote the accuracy of the molecularsubtype classification. Transcript cluster expression was summarized atthe gene level using the median luminescence for the transcript clustersassociated with each gene. Gene expression data were normalized at thegene level and across the whole cohort using median expression levels.Normalized data were used as an input for the random forest classifierdefined in the R Package CMS Classifier to label consensus molecularsubtypes.

The output from the CMS Classifier includes four values, each is aposterior probability of how likely a sample is associated with CMS1-4.CMS1 comprises tumors with increased microsatellite instability (MS I-H)and signatures associated with immune infiltration. FIG. 4B provides 25exemplary colorectal neoplasm molecular subtype biomarker genes usefulfor identification of colorectal cancer subtype CMS1. CMS2-4 areassociated with canonical, metabolic, or mesenchymal gene expressionsignatures, respectively. Based on the CMS classifier, 14 out of 117(12%) of individuals were classified as CMS1, 100 out of 117 (85%) wereclassified as CMS2-4 (canonical, metabolic, and mesenchymal), and 3 outof 117 (3%) were classified as mixed CMS1/CMS2 (FIG. 5).

Example 7: Human Stool Sample Procurement, Extraction & Measurement

Human Stool Collection: Patients were asked to defecate into a bucketthat fit over a toilet seat and the resulting samples were picked up bya courier and transported to the Digestive Diseases Research Core Centerat the Washington University School of Medicine (Saint Louis, Mo.). Thestool was aliquoted into 50 mL conical tubes and stored at −80° C. Fromthere, the samples were transported on dry ice to BioGenerator Labs(Saint Louis, Mo.) where they were stored in a −80° C. freezer untilextraction. The patients used for this collection were consented by theWashington University School of Medicine. Washington University Schoolof Medicine Internal Review Board also provided ethical oversight forthis collection.

Human Sample Types: Stool samples were obtained from 6 patients withcolorectal cancer (stage I-IV), 4 patients with pre-cancerous adenomas,and 14 patients with negative findings on a colonoscopy, resulting in 24aggregate samples. These samples were derived from Human StoolCollection at both Kharkiv National Medical University and WashingtonUniversity School of Medicine. The sample labels were identified andmatched in a manner consistent with criteria outlined previously fromHuman Sample Types.

Total Nucleic Acid Extraction: seRNA was extracted from the samples in amanner consistent with methods outlined previously for Total NucleicAcid Extraction, including DNAse Treatment, and the quality of the seRNAwas analyzed in a manner consistent with methods outlined in ExtractionResults.

Example 8: Analysis of RNA Transcripts

Library Preparation: Libraries of the seRNA were generated using anIllumina Targeted RNA Custom Panel that consisted of 398 customamplicons. Library preparation relied on the steps of initial synthesisof cDNA using ProtoScript II Reverse Transcriptase (Illumina),hybridization of the oligo pool to the targeted seRNA, extension of theoligos using Illumina reagents (AM1, ELM4, RSB, UB1), and amplificationthrough polymerase chain reaction (PCR). Total mass input ranged from200-400 ng and the number of PCR cycles used ranged from 26-28×. Afterlibrary amplification, the cDNA capture was cleaned using Illuminareagents (RSB, AMPure, XP bead EtOh). Library preparations were analyzedfor quantity and quality using Agilent BioAnalyzer and QubitFluorometric Quantitation (Thermo Fisher). All samples described in thisanalysis passed initial quality check and were eligible fornext-generation sequencing.

Sequencing: Unique indices were used for individual samples to allow forpooling of library preparations and multiplexing of all samples into thesame flow cell on an Illumina NextSeq System. All 24 samples were pooledacross one lane in a mid-output flow cell (Illumina). The first 150base-pairs on each end of a read were sequenced (2×150) and sequencedreads were appended to output FASTQ files. Quality check of the FASTQfiles showed that 19 samples had adequate total reads and adequatequality for bioinformatic analysis.

Alignment: After sequencing, custom primer sequences were trimmed fromthe sequence and trimmed reads were aligned to the most currentreference genome (GRCh38). Transcript expression was obtained bycalculating the average coverage across loci. Transcript expressionswere normalized by average coverage for two housekeeping genes (GAPDHand ACTB).

Example 9: Biological Replicates on Various Platforms

Four samples were evaluated on both microarray and sequencing. Linearregression of 398 transcripts across platforms showed moderatereproducibility (Pearson's r range=0.48-0.63). Sequencing showedincreased resolution relative to microarray as evidenced by range ofsignal for transcripts with low luminescence (FIG. 6).

Example 10: Hierarchical Clustering Analysis Using seRNA

Unsupervised principal component analysis (PCA) was performed on RNAsequencing data for all 13 unique samples. Clustering was observedamongst patients with CRC, patients with adenomas, and patients with noneoplastic findings. Samples from patients with cancer demonstrated thelargest variation and separation from other patient populations, whereassamples from patients with no neoplastic findings demonstrated morenarrow clustering (FIG. 7).

Example 11: Evaluation of Sequencing Variants Using seRNA

Variant Calling & Annotation: Integrative Genomics Viewer was used toidentify variants implicated in CRC tumorigenesis. The amplicon panelcovered about 3% of the genomic space for the 398 captured genes.Exemplary driver mutations are shown in FIG. 9. As shown in FIG. 8, weidentified several potential driver mutations. These mutations includeda missense mutation in APC (13% Variant Allele Frequency (VAF)) in apatient with high risk adenomas, a missense mutation in SMAD4 (17% VAF)in a patient with high risk adenomas, a 3′ deletion in the regulatoryregion of MAPK3 (7% VAF) in a patient with stage I CRC, a missensemutation in PIK3CA (12% VAF) in a patient with no findings on acolonoscopy, a missense mutation in KRAS (3% VAF) in a patient with highrisk adenomas, and a missense mutation in CDH1 (2% VAF) in a patientwith high risk adenomas (FIG. 8).

Example 12: Human Stool Sample Procurement

Human Stool Collection: Stool samples were obtained by the DigestiveDisease Research Core Center (DDRCC) at the Washington University Schoolof Medicine (St. Louis, Mo.). All patients were sent a stool samplecollection kit by mail and returned the kit via courier to the DDRCC.Clinical data (e.g., demographic information, colonoscopy results, etc.)were collected by the DDRCC. Each sample was tested for blood in thestool using a commercially available fecal immunochemical test (FIT)(Polymedco, OC-Light S FIT) prior to being frozen at −80° C. Eachpatient recruited for the study had a colonoscopy performed and thosewith positive findings underwent biopsy and subsequent histopathologicreview to determine neoplastic classification. Adenoma classificationwas stratified based on histopathology (benign vs. precancerous), numberof polyps, size of polyps, and differentiation. Cancer classificationwas stratified based on the American Joint Committee on Cancer (AJCC) 7TNM system. If the patient had no findings during the colonoscopy, he orshe was labeled as healthy.

Human Sample Types: In total, stool samples from 275 individuals werecollected for this study. Sequencing data, a FIT, demographicinformation (i.e., gender, age, ethnicity, smoking status, and familyhistory), and colonoscopy results with histopathology information, ifapplicable, were obtained for all patients. In the study, 11 patientshad CRC (stage I-IV), 26 patients had high-risk adenomas (HRAs), 37patients had medium-risk adenomas (MRAs), 61 patients had low-riskadenomas (LRAs), 50 patients had benign polyps, and 90 patients had nofindings on a colonoscopy. Patient type, demographics, and processinginformation are summarized in FIG. 10. Healthy individuals were patientswith no findings on a colonoscopy and no history of colorectal cancer,inflammatory bowel disease, celiac disease, irritable bowel syndrome,diarrhea within the last 20 days or any other gastrointestinal disease.Benign polyp patients provided a stool sample prior to undergoing acolonoscopy where the physician detected a polyp that was deemed to bebenign via a subsequent biopsy and histological evaluation. Diseasedindividuals were patients diagnosed with colorectal cancer orprecancerous adenomas. Colorectal cancer patients had been diagnosedwith stage I-stage IV colorectal cancer via colonoscopy and subsequentbiopsy within the last month and had not yet received any post-biopsytreatment, which can include chemotherapy, radiation, and/or surgery.Precancerous adenoma patients (HRAs, MRAs, and LRAs) provided a stoolsample prior to undergoing a colonoscopy where the physician detected apolyp that was deemed to be precancerous via a subsequent biopsy andhistological evaluation. Stratification of adenoma risk was based onsize of the polyp, number of polyps, extent of dysplasia, and cellularmorphology. The patient population was enriched for colorectal cancerpatients, but the remainder of the samples were representative of anasymptomatic screening population. The patients used for this collectionwere consented by the Washington University School of Medicine. TheWashington University School of Medicine Internal Review Board providedethical oversight for this collection ORB #20111107).

Separation into Training & Testing Sets: 154 prospectively collectedstool samples were used as a training set and 110 prospectivelycollected stool samples were used as a hold-out test set. 11retrospectively collected stool samples from CRC patients were alsoincluded in the hold out test set. The training set and hold out testset were evaluated for categorical, demographic, and handlingdifferences using a t-test (population means) or z-test (populationfrequencies), and significance was indicated if the p-value was lessthan 0.05. There were two statistically significant differences betweenthe characteristics of the training set and the hold out test set.First, retrospectively collected samples (i.e., samples from patientswith CRC) were not included in the training set. Second, the hold outtest set had different processing quality relative to the training set.Specifically, there was a reduction in the average stool input used forstool-derived eukaryotic RNA extraction (12.9 grams vs. 12.0 grams;p-value=0.03), there was a reduction in the average stool-derivedeukaryotic RNA concentration (168.6 ng/uL vs. 56.1 ng/uL; p-value<0.01), and there was a reduction in average library preparationfragment size (200.6 base pairs vs. 192.2 base pairs; p-value <0.01)(FIG. 10).

Example 13: Development of a Custom Capture Panel

Panel Transcripts: A custom capture panel of 639 amplicons was developedfor library preparation in the Illumina DesignStudio. The custom captureprobes were associated with 408 transcripts, which were selected usingpreviously conducted research and the literature.

Microarray Transcripts: Transcripts were selected based on a microarrayexperiment. For this experiment, total seRNA was extracted from stoolsamples and expression was assessed using the Affymetrix HumanTranscriptome Array 2.0 (Thermo Fisher Scientific, Waltham, Mass.).Microarray expression profiles derived from 177 patients with CRC orprecancerous adenomas (diseased cohort) were compared to expressionprofiles from 88 patients with no findings on a colonoscopy (healthycohort). 214 transcripts were identified as being differential expressed(p<0.03) and were selected for the capture panel.

NanoString Transcripts: Transcripts were selected based on a NanoStringexperiment. For this experiment, total seRNA was extracted from stoolsamples and expression was assessed using the nCounter® PanCancerPathways Panel (NanoString, Seattle, Wash.) and the nCounter® PanCancerProgression Panel (NanoString, Seattle, Wash.). NanoString expressionprofiles derived from 59 patients with CRC or precancerous adenomas(diseased cohort) were compared to expression profiles from 26 patientswith no findings on a colonoscopy (healthy cohort). 123 transcripts wereidentified as being differentially expressed and were selected for thecapture panel.

Other Transcripts: The literature was evaluated for additionaltranscripts implicated in CRC. This included searching GeneCards,ClinVar, Catalogue of Somatic Mutations in Cancer (COSMIC), ClinicalInterpretations of Variants in Cancer (CIViC), the Colorectal CancerSubtyping Consortium classifier, and other pertinent studies. 71transcripts were selected for the custom capture panel using theseliteratures.

Example 14: Human Nucleic Acid Extraction

Total Nucleic Acid Extraction: Each stool sample was placed into a 50 mLconical tube. Approximately 6,000-25,000 mg of stool was added to eachtube. An additional 20-40 mL of solution was added to each tube. Thissolution contained a mixture of 10 mM Trizma base (Sigma-Aldrich, St.Louis, Mo.), 1 mM EDTA (Sigma Aldrich) with 0.05% Tween-20(Sigma-Aldrich) and 0.0002% RNase Inhibitor (Sigma-Aldrich) at pH 7.5.The solution was centrifuged at 1000 rpm at 4° C. for 10 minutes and thesupernatant was discarded. Approximately 4-10 mL of EasyMag® LysisBuffer (bioMerieux, Durham, N.C.) was added to the pellet and the pelletwas re-suspended into solution. The solution was centrifuged at2500-3500 rpm at 20-25° C. for 10-15 minutes. During the differentialcentrifugation, the solution separated into three layers. The bottomlayer included solid cellular debris, the middle layer was a hydrophiliclayer enriched for human nucleic acid, and the top layer was ahydrophobic lipid layer. The top two layers were transferred to a new 15mL conical tube and the solution was again centrifuged at 2500 rpm at20-25° C. for 15 minutes. The result from this centrifugation step wasseparation into three layers: the bottom layer was solid cellulardebris, the middle layer was a hydrophilic layer enriched for humannucleic acid, and the top layer was a hydrophobic lipid layer. To screenlarge debris from the solution, a 10 uL pipette tip was placed onto a 1mL pipette tip and 2 mL of the hydrophilic layer was pipetted from the15 mL tube and transferred to an EasyMag® Disposable cartridge(bioMerieux). Additionally, 50 uL of EasyMag® Magnetic Silica(bioMerieux) was added to the cartridge. The beads were mixed into thesolution for 0.5-1 minute using a pipette. The nucleic acids, which werebound to the beads, were eluted into a buffer solution using theSpecific A Protocol according to the manufacturer's directions. Thevolume of the eluted nucleic acids was 70 uL. This nucleic acid solutionwas pipetted into a 1.5 mL tube and placed on ice. The same EasyMag®Disposable cartridges (bioMerieux) that were used in the previous stepwere then reloaded with an additional 2 mL of the hydrophilic layer fromthe same solution in the 15 mL tube used previously using the sametechnique to screen out large debris. An additional 20 uL of EasyMag®Magnetic Silica (bioMerieux) was added to the cartridge. The beads weremixed into the solution for 0.5-1 minute using a pipette. As describedabove, the nucleic acids, which were bound to the beads, were elutedinto a buffer solution using the Specific A Protocol according to themanufacturer's directions. The volume of the eluted nucleic acids was 70uL. This nucleic acid solution was pipetted into the original 1.5 mLtube that already contained first 70 uL eluate and the combined solutionwas placed on ice. An additional 2 mL of the hydrophilic layer from thesame 15 mL solution previously used was added to a new EasyMag®Disposable cartridge (bioMerieux) using the same technique to screen outlarge debris. Additionally, 20 uL of EasyMag® Magnetic Silica(bioMerieux) was added to the cartridge. The beads were mixed into thesolution for 0.5-1 minute using a pipette. The nucleic acids, which werebound to the beads, were eluted into a buffer solution using theSpecific A Protocol according to the manufacturer's directions. Thevolume of the eluted nucleic acids was 70 uL. This nucleic acid solutionwas pipetted into the 1.5 mL tube containing the first two eluates andthe combined solution was placed on ice. The same EasyMag® Disposablecartridges (bioMerieux) that were used in the previous step were thenreloaded with an additional 2 mL of the hydrophilic layer from the samesolution in the 15 mL tube used previously using the same technique toscreen out large debris. An additional 20 uL of EasyMag® Magnetic Silica(bioMerieux) was added to the cartridge. The beads were mixed into thesolution for 0.5-1 minute using a pipette. As described above, thenucleic acids, which were bound to the beads, were eluted into a buffersolution using the Specific A Protocol according to the manufacturer'sdirections. The volume of the eluted nucleic acids was 70 uL. Thisnucleic acid solution was pipetted into the original 1.5 mL tube thatalready contained the first three 70 uL eluates and the combinedsolution was placed on ice.

DNase Treatment: The 280 uL solution was treated withBaseline-Zero-DNase (Epicenter) at 35-40° C. for 20-40 minutes. A 1-2 mLaliquot of EasyMag® Lysis Buffer was added to the DNase treated solutionand the sample was transferred to a new EasyMag® Disposable cartridge.The entire solution was added to the new cartridge along with 85 uL ofEasyMag® Magnetic Silica. The nucleic acids, which were bound to thebeads, were eluted into a buffer solution using the EasyMag® GenericProtocol according to the manufacturer's directions. The volume of theeluted nucleic acids was 25 uL. This nucleic acid solution was pipettedinto a 1.5 mL tube and stored at −80° C.

Example 15: Measurement of Human Nucleic Acid Levels in Human StoolSamples

Extraction Results: 1-2 uL of each of the samples extracted above wasevaluated for total nucleic acid and RNA integrity using the Agilent2100 Bioanalyzer. The samples were analyzed qualitatively andquantitatively. Electrophoretic analysis was used to check the qualityof the extracted RNA. The electrophoresis file was read by comparing thebands for each sample to the bands represented by the size markers inthe RNA ladder and identifying the 18S and 28S ribosomal RNA (rRNA)bands. The rRNA bands are the two large and prominent bands around the2,000-nucleotide marker on the standardization ladder. Qualitatively,adequate banding and darker band intensities indicated that ample intactnucleic acid was available for further analysis such as microarraysequencing, polymerase chain reaction (PCR), nucleic acid sequencing,molecular barcoding, amplicon sequencing, or probe-capture. Theelectropherogram is a graphical representation for each electrophoresisfile with a quantification of the RNA Integrity Number (RIN), total RNAmass, and total rRNA mass. Quantitatively, the larger the RIN, the moretotal RNA mass, and the more total rRNA mass, the higher the likelihooda sample would be useful for further analysis such as microarraysequencing, polymerase chain reaction (PCR), nucleic acid sequencing,molecular barcoding, amplicon sequencing, or probe-capture. Samples werealso evaluated for RNA concentration using the Qubit 4.0 Fluorometer.RNA concentration is determined by quantification of fluorescencegenerated by Qubit assay components, which selectively bind to RNApresent in eluates. Quantitatively, the higher the RNA concentration,the higher the likelihood a sample would be useful for further analysissuch as microarray sequencing, polymerase chain reaction (PCR), nucleicacid sequencing, molecular barcoding, amplicon sequencing, orprobe-capture.

Example 16: Analysis of RNA Transcripts

Library Preparation: Libraries of the seRNA were generated using anIllumina Targeted RNA Custom Panel that consisted of 639 customamplicons. Library preparation relied on the steps of initial synthesisof cDNA using ProtoScript II Reverse Transcriptase (Illumina, San Diego,Calif.), hybridization of the oligo pool to the targeted seRNA,extension of the oligos using Illumina reagents (AM1, ELM4, RSB, UB1),and amplification through polymerase chain reaction (PCR). Total massinput ranged from 200-400 ng and the number of PCR cycles used rangedfrom 28×-30×. After library amplification, the cDNA capture was cleanedusing Illumina reagents (RSB, AMPure, XP bead EtOH). Librarypreparations were analyzed for quantity and quality using the Agilent2100 BioAnalyzer and the Qubit 4.0 Fluorometer (Thermo Fisher). Allsamples described in this analysis passed initial quality check and wereeligible for downstream analysis.

Sequencing Analysis: Unique indices were used for individual samples toallow for pooling of library preparations and multiplexing of samplesinto flow cells on an Illumina NextSeq 550 System. A PhiX spike-in wasused for quality control. The 275 samples were pooled across 8individual high-output flow cell runs (IIlumina). Up to 150 base-pairson each end of a read were sequenced (2×150) and sequenced reads wereappended to output FASTQ files. Quality check of the FASTQ files showedthat all 275 samples had adequate total reads (>100,000) and adequatequality for bioinformatic analysis.

Alignment: After sequencing, custom primer sequences were trimmed fromthe read and aligned to the most current reference genome (GRCh38) viaHISAT2.3.0. Transcript expression was obtained by calculating theaverage coverage across loci. For each transcript, raw ampliconexpression was normalized to GAPDH, an internal housekeeping gene, suchthat reported expression equates to amplicon read count per millionmapped-GAPDH reads.

Transcript Selection: Normalized expression of 639 amplicons wasevaluated for all samples in the training set (n=154 samples). Of these639 amplicons, 48 amplicons were not expressed in any sample and anadditional 71 amplicons were not expressed in >95% of all samples; theseamplicons were eliminated from the analysis. For the remainingamplicons, a bootstrap analysis was performed by splitting the trainingset into 100 different 9:1 splits, whereby each split was assessed forinformative amplicons. An amplicon was considered informative if theabsolute log₂ fold-change was greater than 1 in both contrast groups(HRAs vs. LRAs, benign polyps, no findings on colonoscopy; MRAs vs.LRAs, benign polyps, no findings on colonoscopy) and the ANOVA betweenthe contrast groups had a p-value <0.05. The transcript selectionprocess is further illustrated in FIG. 11A. In total, there were 40amplicons from 29 genes identified as informative in at least 1 of the100 splits (FIG. 11B). If an amplicon was deemed informative in at least33% of all bootstrapped splits, it was considered differentiallyexpressed and eligible as a feature for model development. There were 10amplicons identified as differentially expressed (informative in atleast 33 of the 100 splits) (FIG. 11B). Raw GAPDH values are considereda measure for total eukaryotic RNA in each sample. It was observed thatraw GAPDH values were elevated in patients with MRAs, HRAs, and CRC,relative to healthy patients (FIG. 12). Demographic features (age,smoking status, previous family history, ethnicity, and gender) werealso considered for model development. Ultimately, the 10 differentiallyexpressed transcripts, raw GAPDH values, and 2 demographic identifiers(age and smoking status) were eligible as features for modeldevelopment.

Example 17: Random Forest Model Development

A random forest model was built using the 154-patient training set andall 13 eligible features. 5,000 decision trees were constructed frombootstrapped training samples; each node split was optimized by GiniImportance; each tree was built until it reached full depth. Althoughspecific embodiments are discussed herein, it will be appreciated thatany suitable model, such as a random forest model using a greater and/orlesser number of decision trees, a greater and/or lesser number ofeligible features, etc. may be generated. Additionally, other types ofmodels, such as a deep learning model or a support vector model might beused with varied parameters. The random forest model used eligiblefeatures, such as differentially expressed transcripts, raw GAPDHvalues, age, and smoking status. Although specific embodiments arediscussed herein, it will be appreciated that any suitable model, suchas a random forest model using all of the informative features and/or aselected subset of the informative features, may be generated.

Output from the model was configured to provide a prediction between 0-1whereby a larger number reflects increased confidence in a neoplastic orpositive finding. A fecal immunochemical test (FIT) was used in someembodiments to alter confidence in a neoplastic or positive finding. Forexample, for a FIT positive sample, the prediction score would increaseto 1. 3-fold internal cross-validation was used to assess training modelperformance. 3-fold internal cross-validation used 3 different 2:1splits whereby a model was built using the larger split and employed onthe smaller split. Receiver operating characteristic (ROC) curves werecreated using model predictions and area under the curve (AUC) was usedto measure model performance. The median ROC curve from the 3 splits wasused to approximate cross-validation performance. ROC curves wereplotted with and without incorporating the FIT feature. For ROC curvesplotted with the FIT feature, a positive FIT forced model prediction toequal 1. In the provided example, internal cross-validation without theFIT feature yielded a ROC AUC of 0.65 for HRAs versus all othercategories (MRAs, LRAs, benign polyps, and no findings on acolonoscopy). In the provided example, internal cross-validation withthe FIT feature yielded a ROC AUC of 0.70 for HRAs versus all othercategories (MRAs, LRAs, benign polyps, and no findings on a colonoscopy)(FIG. 13).

Hold Out Test Set: A final random forest model was built using all 154samples within the training set. For the generated model, the mostinfluential features as measured by Gini Importance were ACY1 andTNFRSF10B (Gini Importance ≥0.13) and the least important feature wasPER3 (Gini Importance <0.05). Raw GAPDH values were the 4th mostimportant feature in building the random forest model (FIG. 14). Thismodel was employed on the 110 prospectively collected stool samples inthe hold out test set. ROC curves were plotted with and without the FITfeature and area under the curve (AUC) was used to measure modelperformance. The model attained a ROC AUC of 0.67 without the FITfeature and a ROC AUC of 0.78 with the FIT feature (FIG. 15).

Model Predictions: Model predictions in the hold out test set werecorrelated with disease severity (FIG. 16). The model output correlationwith disease severity was a direct reflection of the biology and notspecifically trained as part of the model. In the foregoing embodiment,feature selection and model input included the use of three categories(HRAs, MRAs, and all others) however, disease subtypes (e.g., subsets ofHRAs) and disease order (e.g., HRAs are more severe than MRAs) were notused as features for model training. Given that model output iscorrelated to disease severity, this permits prospective identificationof specific subtypes and severity of disease using model output.Further, altering model parameters to provide model with diseaseseverity information improved stratification of positive and negativefindings.

Downsampling Analysis: To understand the extent of model training,downsampling fractions of the 154 samples in the training set wereselected and performance was assessed using the hold out test set. Thedownsampling fractions ranged from 30% to 100% with 10% increments. Foreach downsampling fraction, feature selection was performed usingbootstrapping, a random forest model was trained using the eligiblefeatures, and the model was employed on the hold out test set. The ROCAUC for the hold out test set was used to assess model performance. Thisprocess was repeated 10 times for each downsampling fraction to reduceselection bias in subsampling, and model performance was assessed withand without incorporating the FIT feature. The downsampling analysisshowed a direct relationship between total number of samples used fortraining and performance on the hold out test set. When excluding theFIT feature, the median ROC AUC for HRAs versus all other categoriesincreased from 0.55 (30% of training data) to 0.67 (100% of trainingdata) (FIG. 17A). When including the FIT feature, median ROC AUC forHRAs versus all other categories increased from 0.72 (30% of trainingdata) to 0.78 (100% of training data) (FIG. 17B).

Final Accuracy: Continuing the above embodiment, the random forest modelwas also employed on the 11 retrospectively collected stool samples fromCRC patients. Output from the model provided a prediction between 0-1and a positive FIT forced model prediction to equal 1. Samples having apositive fecal immunochemical test (FIT+) or a positive model prediction(Model+) were considered positive and all other samples were considerednegative. A ROC curve was plotted whereby only CRC samples wereconsidered positive and other categories (HRAs, MRAs, LRAs, benignpolyps, and no findings on a colonoscopy) were considered negative.Using all 121 samples in this supplemented hold out test set, this modelattained a ROC AUC of 0.94. A separate ROC curve was plotted whereby CRCand HRA samples were considered positive and all other categories (MRAs,LRAs, benign polyps, and no findings on a colonoscopy) were considerednegative. Using all 121 samples in this supplemented hold out test set,this model attained a ROC AUC of 0.87 (FIG. 18). A point on the ROCcurve that maximized accuracy was selected to calculate sensitivity andspecificity. At this point, the model demonstrated 91% sensitivity forCRC (n=11 samples) and a 73% sensitivity for HRAs (n=11 samples) at an89% specificity (n=99 samples) (FIG. 18).

Extrapolation to Screening Population: To attain a better approximationof ultimate model performance, the accuracy profile observed on thesupplemented hold out test set was extrapolated to the relativefrequencies expected in a prospective screening population. ROC curvesas described above were plotted to show model performance. Whenweighting cancer and HRAs to expected prevalence in a prospectivescreening population the model attained a ROC AUC of 0.80 for CRC andHRA samples versus all other categories (FIG. 19). Extrapolation ofresults onto a prospective screening population also enables thecalculation of the blended sensitivity for neoplastic findings, negativepredictive value (NPV), and positive predictive value (PPV). Thisextrapolated accuracy profile demonstrated a blended sensitivity for CRCand HRAs of 74%, a positive predictive value of 37%, and a negativepredictive value of 98% (FIG. 19).

1. A method of detecting colorectal neoplasia in a subject, the methodcomprising: a) measuring the level of expression of 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28 or 29 stool-derived eukaryotic RNA biomarkers selected from thebiomarkers listed in Table 1 or Table 2 or a combination of Table 1 andTable 2 in eukaryotic nucleic acid extracted from a stool sample fromthe subject; b) comparing the measured expression level of the 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28 or 29 stool-derived eukaryotic RNA biomarkers in thestool sample with the measured expression level of the 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, or 29 stool-derived eukaryotic RNA biomarkers in a control,wherein a difference in the measured expression level of the 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28 or 29 stool-derived eukaryotic RNA biomarkers in thestool sample relative to the measured expression level of the 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28 or 29 stool-derived eukaryotic RNA biomarkers in thecontrol indicates that the subject has colorectal neoplasia.
 2. Themethod of claim 1, wherein the subject is human.
 3. The method of claim1, wherein the colorectal neoplasia is selected from the groupconsisting of colorectal cancer, high-risk adenoma, medium-risk adenoma,and low-risk adenoma.
 4. (canceled)
 5. The method of claim 1, whereinthe nucleic acid comprises RNA, total RNA, mRNA, seRNA, tRNA, rRNA,ncRNA, smRNA, or snoRNA, or a combination of any of RNA, total RNA,mRNA, seRNA, tRNA, rRNA, ncRNA, smRNA, or snoRNA.
 6. The method of claim1, wherein the expression level is measured by nucleic acid sequencing,microarray sequencing, molecular barcoding, amplicon sequencing, probecapture, polymerase chain reaction (PCR), ddPCR, dPCR, RT-PCR, orRT-qPCR.
 7. The method of claim 1, further comprising determining thesubject's demographic information.
 8. The method of claim 1, furthercomprising administering a fecal immunochemical test (FIT) to thesubject.
 9. A method of selecting a clinical plan for a subject havingor at risk for colorectal neoplasia, the method comprising: a) measuringthe level of expression of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29stool-derived eukaryotic RNA biomarkers selected from the biomarkerslisted in Table 1 or Table 2 or a combination of Table 1 and Table 2 ineukaryotic nucleic acid present in a stool sample from the subject; b)comparing the measured expression level of the 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28 or 29 stool-derived eukaryotic RNA biomarkers in the stool samplewith the measured expression level of the 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28or 29 stool-derived eukaryotic RNA biomarkers in a control, wherein adifference in the measured expression level of the 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28 or 29 stool-derived eukaryotic RNA biomarkers in the stool samplerelative to the measured expression level of the 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28 or 29 stool-derived eukaryotic RNA biomarkers in the controlindicates that the subject has or is at risk for colorectal neoplasia;c) administering a diagnostic procedure or a treatment or a combinationof a diagnostic procedure and a treatment.
 10. The method of claim 9,wherein the subject is a human.
 11. The method of claim 9, wherein thecolorectal neoplasia is selected from the group consisting of colorectalcancer, high-risk adenoma, medium-risk adenoma, and low-risk adenoma.12. (canceled)
 13. The method of claim 9, wherein the nucleic acidcomprises RNA, total RNA, mRNA, seRNA, tRNA, rRNA, ncRNA, smRNA, orsnoRNA, or a combination of any of RNA, total RNA, mRNA, seRNA, tRNA,rRNA, ncRNA, smRNA, or snoRNA.
 14. The method of claim 9, wherein theexpression level is measured by nucleic acid sequencing, microarraysequencing, molecular barcoding, amplicon sequencing, probe capture,polymerase chain reaction (PCR), ddPCR, dPCR, RT-PCR, or RT-qPCR. 15.The method of claim 9, further comprising determining the subject'sdemographic information.
 16. The method of claim 9, further comprisingadministering a fecal immunochemical test (FIT) to the subject.
 17. Themethod of claim 9, wherein the clinical plan comprises a diagnosticprocedure or a treatment.
 18. The method of claim 9, wherein thediagnostic procedure comprises a colonoscopy.
 19. (canceled) 20.(canceled)
 21. (canceled)
 22. (canceled)
 23. (canceled)
 24. (canceled)25. (canceled)
 26. A method of treating a colorectal neoplasia in asubject, the method comprising: a) determining that a stool sample fromthe patient expresses elevated levels of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29stool-derived eukaryotic RNA biomarkers selected from the biomarkerslisted in Table 1 or Table 2 or a combination of Table 1 and Table 2 ineukaryotic nucleic acid present in a stool sample from the subject; andb) administering to the subject a colonoscopy, surgery, chemotherapy,radiation therapy, targeted therapy, or immunotherapy.
 27. A method ofdetecting colorectal neoplasia in a subject, the method comprising: a)measuring the variant allele frequency of one or more variant biomarkergenes selected from the biomarker genes listed in Table 3 in eukaryoticnucleic acid extracted from a stool sample from the subject; b)comparing the measured variant allele frequency of the one or morevariant biomarker genes in the stool sample with the measured variantallele frequency of the one or more variant biomarker genes in acontrol, wherein a difference in the variant allele frequency of the oneor more variant biomarker genes relative to the variant allele frequencyof the one or more variant biomarker genes in the control indicates thatthe subject has or is at risk for colorectal cancer.
 28. A method ofdetermining whether a subject has or is at risk for colorectal cancer,the method comprising: a) measuring the variant allele frequency of oneor more variant biomarker genes selected from the biomarker genes listedin Table 3 in eukaryotic nucleic acid extracted from a stool sample fromthe subject; b) comparing the measured variant allele frequency of theone or more variant biomarker genes in the stool sample with themeasured variant allele frequency of the one or more variant biomarkergenes in a control, wherein a difference in the variant allele frequencyof the one or more variant biomarker genes relative to the variantallele frequency of the one or more variant biomarker genes in thecontrol indicates that the subject has or is at risk for colorectalcancer.
 29. (canceled)
 30. (canceled)
 31. (canceled)
 32. (canceled) 33.(canceled)
 34. (canceled)
 35. (canceled)
 36. (canceled)
 37. A method ofdetecting a molecular subtype of colorectal cancer in a subject, themethod comprising: a) measuring the level of expression of two or morebiomarker genes selected from any of the colorectal neoplasm molecularsubtype biomarker genes listed in Table 4 in eukaryotic nucleic acidextracted from a stool sample from the subject; b) comparing themeasured expression level of the two or more colorectal neoplasmmolecular subtype biomarker genes in the biological sample with themeasured expression level of the two or more colorectal neoplasmmolecular subtype biomarker genes in a control, wherein a difference inthe measured expression level of the two or more colorectal neoplasmmolecular subtype biomarker genes in the biological sample with themeasured expression level of the two or more colorectal neoplasmmolecular subtype biomarker genes relative to the two or more colorectalneoplasm molecular subtype biomarker genes in the control indicates themolecular subtype of colorectal cancer.
 38. (canceled)
 39. (canceled)40. (canceled)
 41. (canceled)
 42. (canceled)
 43. (canceled) 44.(canceled)
 45. (canceled)
 46. (canceled)
 47. (canceled)
 48. (canceled)49. A method comprising: a) determining that a stool sample from apatient comprises elevated levels of two or more stool-derivedeukaryotic biomarkers selected from the biomarkers listed in Table 1 orTable 2 or a combination of Table 1 and Table 2 or Table 3 or Table 4 ineukaryotic nucleic acid present in a stool sample from the subjectrelative to a control; b) based on that determination, determining thepatient is at risk for a colorectal neoplasia; and c) administering acolonoscopy to the patient.
 50. The method of claim 49, furthercomprising step of providing a stool sample.